A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan.

Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.

Antisense Oligonucleotide-Based Therapy for Neuromuscular Disease.

Neuromuscular disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy are neurodegenerative genetic diseases characterized primarily by muscle weakness and wasting. Until recently there were no effective therapies for these conditions, but antisense oligonucleotides, a new class of synthetic single stranded molecules of nucleic acids, have demonstrated promising experimental results and are at different stages of regulatory approval. The antisense oligonucleotides can modulate the protein expression via targeting hnRNAs or mRNAs and inducing interference with splicing, mRNA degradation, or arrest of translation, finally, resulting in rescue or reduction of the target protein expression. Different classes of antisense oligonucleotides are being tested in several clinical trials, and limitations of their clinical efficacy and toxicity have been reported for some of these compounds, while more encouraging results have supported the development of others. New generation antisense oligonucleotides are also being tested in preclinical models together with specific delivery systems that could allow some of the limitations of current antisense oligonucleotides to be overcome, to improve the cell penetration, to achieve more robust target engagement, and hopefully also be associated with acceptable toxicity. This review article describes the chemical properties and molecular mechanisms of action of the antisense oligonucleotides and the therapeutic implications these compounds have in neuromuscular diseases. Current strategies and carrier systems available for the oligonucleotides delivery will be also described to provide an overview on the past, present and future of these appealing molecules.

Vitamin D in corticosteroid-naïve and corticosteroid-treated Duchenne muscular dystrophy: what dose achieves optimal 25(OH) vitamin D levels?

AIM: Assessment of the efficacy of vitamin D replenishment and maintenance doses required to attain optimal levels in boys with Duchenne muscular dystrophy (DMD). METHOD: 25(OH)-vitamin D levels and concurrent vitamin D dosage were collected from retrospective case-note review of boys with DMD at the Dubowitz Neuromuscular Centre. Vitamin D levels were stratified as deficient at <25 nmol/L, insufficient at 25-49 nmol/L, adequate at 50-75 nmol/L and optimal at >75 nmol/L. RESULT: 617 vitamin D samples were available from 197 boys (range 2-18 years)-69% from individuals on corticosteroids. Vitamin D-naïve boys (154 samples) showed deficiency in 28%, insufficiency in 42%, adequate levels in 24% and optimal levels in 6%. The vitamin D-supplemented group (463 samples) was tested while on different maintenance/replenishment doses. Three-month replenishment of daily 3000 IU (23 samples) or 6000 IU (37 samples) achieved optimal levels in 52% and 84%, respectively. 182 samples taken on 400 IU revealed deficiency in 19 (10%), insufficiency in 84 (47%), adequate levels in 67 (37%) and optimal levels in 11 (6%). 97 samples taken on 800 IU showed deficiency in 2 (2%), insufficiency in 17 (17%), adequate levels in 56 (58%) and optimal levels in 22 (23%). 81 samples were on 1000 IU and 14 samples on 1500 IU, with optimal levels in 35 (43%) and 9 (64%), respectively. No toxic level was seen (highest level 230 nmol/L). CONCLUSIONS: The prevalence of vitamin D deficiency and insufficiency in DMD is high. A 2-month replenishment regimen of 6000 IU and maintenance regimen of 1000-1500 IU/day was associated with optimal vitamin D levels. These data have important implications for optimising vitamin D dosing in DMD.

Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling.

Mitochondrial Ca(2+) uptake has key roles in cell life and death. Physiological Ca(2+) signaling regulates aerobic metabolism, whereas pathological Ca(2+) overload triggers cell death. Mitochondrial Ca(2+) uptake is mediated by the Ca(2+) uniporter complex in the inner mitochondrial membrane, which comprises MCU, a Ca(2+)-selective ion channel, and its regulator, MICU1. Here we report mutations of MICU1 in individuals with a disease phenotype characterized by proximal myopathy, learning difficulties and a progressive extrapyramidal movement disorder. In fibroblasts from subjects with MICU1 mutations, agonist-induced mitochondrial Ca(2+) uptake at low cytosolic Ca(2+) concentrations was increased, and cytosolic Ca(2+) signals were reduced. Although resting mitochondrial membrane potential was unchanged in MICU1-deficient cells, the mitochondrial network was severely fragmented. Whereas the pathophysiology of muscular dystrophy and the core myopathies involves abnormal mitochondrial Ca(2+) handling, the phenotype associated with MICU1 deficiency is caused by a primary defect in mitochondrial Ca(2+) signaling, demonstrating the crucial role of mitochondrial Ca(2+) uptake in humans.

Characterization of recessive RYR1 mutations in core myopathies.

We have characterized at the molecular level, three families with core myopathies carrying apparent recessive mutations in their RYR1 gene and studied the pharmacological properties of myotubes carrying endogenous mutations as well as the properties of mutant channels expressed in HEK293 cells. The proband of family 1 carried p.Ala1577Thr+p.Gly2060Cys in trans, having inherited a mutation from each parent. Immunoblot analysis of proteins from the patient's skeletal muscle revealed low levels of ryanodine receptor (RyR1) but neither substitution alone or in combination affected the functional properties of RyR1 channels in a discernable way. Two affected siblings in family 2 carried p.Arg109Trp+p.Met485Val substitutions in cis, inherited from the unaffected father. Interestingly, both affected siblings only transcribed the mutated paternal allele in skeletal muscle, whereas the maternal allele was silent. Single-channel measurements showed that recombinant, mutant RyR1 channels carrying both substitutions lost the ability to conduct Ca2+. In this case as well, low levels of RyR1 were present in skeletal muscle extracts. The proband of family 3 carried p.Ser71Tyr+p.Asn2283His substitutions in trans. Recombinant channels with Asn2283His substitution showed an increased activity, whereas recombinant channels with p.Ser71Tyr+p.Asn2283His substitution lost activity upon isolation. Taken together, our data suggest major differences in the ways RYR1 mutations may affect patients with core myopathies, by compromising RyR1 protein expression, stability and/or activity.

Magnetic resonance imaging of muscle in congenital myopathies associated with RYR1 mutations.

Mutations in the skeletal muscle ryanodine receptor (RYR1) gene are associated with a wide range of phenotypes, comprising central core disease and distinct subgroups of multi-minicore disease. We report muscle MRI findings of 11 patients from eight families with RYR1 mutations (n=9) or confirmed linkage to the RYR1 locus (n=2). Patients had clinical features of a congenital myopathy with a wide variety of associated histopathological changes. Muscle MR images showed a consistent pattern characterized by (a) within the thigh: selective involvement of vasti, sartorius, adductor magnus and relative sparing of rectus, gracilis and adductor longus; (b) within the lower leg: selective involvement of soleus, gastrocnemii and peroneal group and relative sparing of the tibialis anterior. Our findings indicate that patients with RYR1-related congenital myopathies have a recognizable pattern of muscle involvement irrespective of the variability of associated histopathological findings. Muscle MRI may supplement clinical assessment and aid selection of genetic tests particularly in patients with non-diagnostic or equivocal histopathological features.