Noninvasive ¹³C-octanoic acid breath test shows delayed gastric emptying in patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive loss of motor neurons. However, ALS has been recognized to also involve non-motor systems. Subclinical involvement of the autonomic system in ALS has been described. The recently developed C-13-octanoic acid breath test allows the noninvasive measurement of gastric emptying. With this new technique we investigated 18 patients with ALS and 14 healthy volunteers. None of the patients had diabetes mellitus or other disorders known to cause autonomic dysfunction. The participants received a solid standard test meal labeled with C-13-octanoic acid. Breath samples were taken at 15-min intervals for 5 h and were analyzed for (CO2)-C-13 by isotope selective nondispersive infrared spectrometry. Gastric emptying peak time (t(peak)) and emptying half time (t(1/2)) were determined. All healthy volunteers displayed normal gastric emptying with a mean emptying t(1/2) of 138 +/- 34 (range 68-172) min. Gastric emptying was delayed (t(1/2) > 160 min) in 15 of 18 patients with ALS. Emptying t(1/2) in ALS patients was 218 +/- 48 (range 126-278) min (p < 0.001). These results are compatible with autonomic involvement in patients with ALS, causing delayed gastric emptying of solids and encouraging the theory that ALS is a multisystem disease rather than a disease of the motor neurons only

U7 snRNAs induce correction of mutated dystrophin pre-mRNA by exon skipping.

Most cases of Duchenne muscular dystrophy are caused by dystrophin gene mutations that disrupt the mRNA reading frame. Artificial exclusion (skipping) of a single exon would often restore the reading frame, giving rise to a shorter, but still functional dystrophin protein. Here, we analyzed the ability of antisense U7 small nuclear (sn)RNA derivatives to alter dystrophin pre-mRNA splicing. As a proof of principle, we first targeted the splice sites flanking exon 23 of dystrophin pre-mRNA in the wild-type muscle cell line C2C12 and showed precise exon 23 skipping. The same strategy was then successfully adapted to dystrophic immortalized mdx muscle cells where exon-23-skipped dystrophin mRNA rescued dystrophin protein synthesis. Moreover, we observed a stimulation of antisense U7 snRNA expression by the murine muscle creatine kinase enhancer. These results demonstrate that alteration of dystrophin pre-mRNA splicing could correct dystrophin gene mutations by expression of specific U7 snRNA constructs

Autologous skeletal muscle derived cells expressing a novel functional dystrophin provide a potential therapy for Duchenne Muscular Dystrophy

Autologous stem cells that have been genetically modified to express dystrophin are a possible means of treating Duchenne Muscular Dystrophy (DMD). To maximize the therapeutic effect, dystrophin construct needs to contain as many functional motifs as possible, within the packaging capacity of the viral vector. Existing dystrophin constructs used for transduction of muscle stem cells do not contain the nNOS binding site, an important functional motif within the dystrophin gene. In this proof-of-concept study, using stem cells derived from skeletal muscle of a DMD patient (mdcs) transplanted into an immunodeficient mouse model of DMD, we report that two novel dystrophin constructs, C1 (ΔR3-R13) and C2 (ΔH2-R23), can be lentivirally transduced into mdcs and produce dystrophin. These dystrophin proteins were functional in vivo, as members of the dystrophin glycoprotein complex were restored in muscle fibres containing donor-derived dystrophin. In muscle fibres derived from cells that had been transduced with construct C1, the largest dystrophin construct packaged into a lentiviral system, nNOS was restored. The combination of autologous stem cells and a lentivirus expressing a novel dystrophin construct which optimally restores proteins of the dystrophin glycoprotein complex may have therapeutic application for all DMD patients, regardless of their dystrophin mutation

A Phase 2 Study of AMO-02 (tideglusib) in Congenital and Childhood Onset Myotonic Dystrophy Type 1 (DM1)

Background: GSK3β is an intracellular regulatory kinase that is dysregulated in multiple tissues in Type 1 myotonic dystrophy (DM1), a rare neuromuscular disorder that manifests at any age. AMO-02 (tideglusib) inhibits GSK3β activity in preclinical models of DM1 and promotes cellular maturation as well as normalizing aberrant molecular and behavioral phenotypes. This Phase 2 study assessed the pharmacokinetics, safety and tolerability, and preliminary efficacy, of AMO-02 in adolescents and adults with Congenital and Childhood-onset DM1. Methods: Sixteen subjects (aged 13 to 34) with Congenital and Childhood-onset DM1 received 12 weeks of single-blind fixed-dose oral treatment with either 400 mg (n=8) or 1000 mg (n=8) of AMO-02 (NCT02858908). Blood samples were obtained for pharmacokinetic assessment. Safety assessments, such as laboratory tests and ECGs, as well as efficacy assessments of syndromal, cognitive and muscular functioning, were obtained. Results: AMO-02 plasma concentrations conformed to a two-compartment model with first-order absorption and elimination, and dose-dependent increases in exposure (area-under-the-curve, or AUC) were observed. AMO-02 was generally safe and well-tolerated. No early discontinuations due to adverse events nor dose adjustments of AMO-02 occurred. The majority of subjects manifested clinical improvement in their CNS and neuromuscular symptoms after 12 weeks of treatment compared to the placebo baseline, with a larger response noted at the 1000 mg/day dose level. AMO-02 exposure (cumulative AUC) was significantly correlated (p<0.01) with change from baseline on several key efficacy assessments. Conclusion: AMO-02 has favorable pharmacokinetic and clinical risk/benefit profiles meriting further study as a potential treatment for Congenital and Childhood-onset DM1

Scapuloperoneal syndrome type Kaeser and a wide phenotypic spectrum of adult-onset, dominant myopathies are associated with the desmin mutation R350P

In 1965, an adult-onset, autosomal dominant disorder with a peculiar scapuloperoneal distribution of weakness and atrophy was described in a large, multi-generation kindred and named ‘scapuloperoneal syndrome type Kaeser' (OMIM #181400). By genetic analysis of the original kindred, we discovered a heterozygous missense mutation of the desmin gene (R350P) cosegregating with the disorder. Moreover, we detected DES R350P in four unrelated German families allowing for genotype-phenotype correlations in a total of 15 patients carrying the same mutation. Large clinical variability was recognized, even within the same family, ranging from scapuloperoneal (n = 2, 12%), limb girdle (n = 10, 60%) and distal phenotypes (n = 3, 18%) with variable cardiac (n = 7, 41%) or respiratory involvement (n = 7, 41%). Facial weakness, dysphagia and gynaecomastia were frequent additional symptoms. Overall and within each family, affected men seemingly bear a higher risk of sudden, cardiac death as compared to affected women. Moreover, histological and immunohistochemical examination of muscle biopsy specimens revealed a wide spectrum of findings ranging from near normal or unspecific pathology to typical, myofibrillar changes with accumulation of desmin. This study reveals that the clinical and pathological variability generally observed in desminopathies may not be attributed to the nature of the DES mutation alone, but may be influenced by additional genetic and epigenetic factors such as gender. In addition, mutations of the desmin gene should be considered early in the diagnostic work-up of any adult-onset, dominant myopathy, even if specific myofibrillar pathology is absen

Design, set-up and utility of the UK facioscapulohumeral muscular dystrophy patient registry

Facioscapulohumeral dystrophy (FSHD) is a rare inherited neuromuscular disease estimated to affect 1/15,000 people. Through basic research, remarkable progress has been made towards the development of targeted therapies. Patient identification, through registries or other means is essential for trial-readiness. The UK FSHD Patient Registry is a patient initiated registry that collects standardised and internationally agreed dataset of self-reported clinical details combined with professionally verified genetic information. It includes four additional questionnaires to capture patient reported outcomes related to pain, quality of life and scapular fixation. Between 2013 and 2015, 518 patients registered 243 males, 241 females with a mean age of 47.8 years. Most of the patients have FSHD type 1 (91.7 %), and weakness of the facial (59.2 %) was the most prevalent symptom at onset, followed by shoulder-girdle muscles (53.3 %) and distal (22.45 %) or proximal lower limb weakness (14.8 %). 85.57 % patients were ambulant or ambulant with assistance at the time of registration, 7.9 % report respiratory insufficiency. The registry has demonstrated utility with the recruitment of patients for a natural history study of infantile onset FSHD, and the longitudinal analysis of patient-related outcomes will provide much-needed baseline information to power future trials. The internationally agreed core dataset enables national registries to participate in a "Global FSHD registry". We suggest that the registry's ability to interoperate with other large datasets will be instrumental for sharing and exploiting data globally

Circulating small RNA signatures differentiate accurately the subtypes of muscular dystrophies: small-RNA next-generation sequencing analytics and functional insights

Muscular dystrophies are a group of rare and severe inherited disorders mainly affecting the muscle tissue. Duchene Muscular Dystrophy, Myotonic Dystrophy types 1 and 2, Limb Girdle Muscular Dystrophy and Facioscapulohumeral Muscular Dystrophy are some of the members of this family of disorders. In addition to the current diagnostic tools, there is an increasing interest for the development of novel non-invasive biomarkers for the diagnosis and monitoring of these diseases. miRNAs are small RNA molecules characterized by high stability in blood thus making them ideal biomarker candidates for various diseases. In this study, we present the first genome-wide next-generation small RNA sequencing in serum samples of five different types of muscular dystrophy patients and healthy individuals. We identified many small RNAs including miRNAs, lncRNAs, tRNAs, snoRNAs and snRNAs, that differentially discriminate the muscular dystrophy patients from the healthy individuals. Further analysis of the identified miRNAs showed that some miRNAs can distinguish the muscular dystrophy patients from controls and other miRNAs are specific to the type of muscular dystrophy. Bioinformatics analysis of the target genes for the most significant miRNAs and the biological role of these genes revealed different pathways that the dysregulated miRNAs are involved in each type of muscular dystrophy investigated. In conclusion, this study shows unique signatures of small RNAs circulating in five types of muscular dystrophy patients and provides a useful resource for future studies for the development of miRNA biomarkers in muscular dystrophies and for their involvement in the pathogenesis of the disorders

Am J Hum Genet

Escobar syndrome is a form of arthrogryposis multiplex congenita and features joint contractures, pterygia, and respiratory distress. Similar findings occur in newborns exposed to nicotinergic acetylcholine receptor (AChR) antibodies from myasthenic mothers. We performed linkage studies in families with Escobar syndrome and identified eight mutations within the γ-subunit gene (CHRNG) of the AChR. Our functional studies show that γ-subunit mutations prevent the correct localization of the fetal AChR in human embryonic kidney–cell membranes and that the expression pattern in prenatal mice corresponds to the human clinical phenotype. AChRs have five subunits. Two α, one β, and one δ subunit are always present. By switching γ to &epsiv; subunits in late fetal development, fetal AChRs are gradually replaced by adult AChRs. Fetal and adult AChRs are essential for neuromuscular signal transduction. In addition, the fetal AChRs seem to be the guide for the primary encounter of axon and muscle. Because of this important function in organogenesis, human mutations in the γ subunit were thought to be lethal, as they are in γ-knockout mice. In contrast, many mutations in other subunits have been found to be viable but cause postnatally persisting or beginning myasthenic syndromes. We conclude that Escobar syndrome is an inherited fetal myasthenic disease that also affects neuromuscular organogenesis. Because γ expression is restricted to early development, patients have no myasthenic symptoms later in life. This is the major difference from mutations in the other AChR subunits and the striking parallel to the symptoms found in neonates with arthrogryposis when maternal AChR auto-antibodies crossed the placenta and caused the transient inactivation of the AChR pathway