Mutations in the DMD gene, encoding the dystrophin protein, are responsible for the dystrophinopathies Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), and X-linked Dilated ...Cardiomyopathy (XLDC). Mutation analysis has traditionally been challenging, due to the large gene size (79 exons over 2.2 Mb of genomic DNA). We report a very large aggregate data set comprised of DMD mutations detected in samples from patients enrolled in the United Dystrophinopathy Project, a multicenter research consortium, and in referral samples submitted for mutation analysis with a diagnosis of dystrophinopathy. We report 1,111 mutations in the DMD gene, including 891 mutations with associated phenotypes. These results encompass 506 point mutations (including 294 nonsense mutations) and significantly expand the number of mutations associated with the dystrophinopathies, highlighting the utility of modern diagnostic techniques. Our data supports the uniform hypermutability of CGA>TGA mutations, establishes the frequency of polymorphic muscle (Dp427m) protein isoforms and reveals unique genomic haplotypes associated with "private" mutations. We note that 60% of these patients would be predicted to benefit from skipping of a single DMD exon using antisense oligonucleotide therapy, and 62% would be predicted to benefit from an inclusive multiexonskipping approach directed toward exons 45 through 55. Hum Mutat 30:1657-1666, 2009.
Myotonic dystrophy (DM) is caused by expanded CTG/CCTG repeats, causing symptoms in skeletal muscle, heart, and central nervous system (CNS). CNS issues are debilitating and include hypersomnolence, ...executive dysfunction, white matter atrophy, and neurofibrillary tangles. Here, we generate RNA-seq transcriptomes from DM and unaffected frontal cortex and identify 130 high-confidence splicing changes, most occurring only in cortex, not skeletal muscle or heart. Mis-spliced exons occur in neurotransmitter receptors, ion channels, and synaptic scaffolds, and GRIP1 mis-splicing modulates kinesin association. Optical mapping of expanded CTG repeats reveals extreme mosaicism, with some alleles showing >1,000 CTGs. Mis-splicing severity correlates with CTG repeat length across individuals. Upregulated genes tend to be microglial and endothelial, suggesting neuroinflammation, and downregulated genes tend to be neuronal. Many gene expression changes strongly correlate with mis-splicing, suggesting candidate biomarkers of disease. These findings provide a framework for mechanistic and therapeutic studies of the DM CNS.
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•DM1 RNA-seq shows mis-splicing of neurotransmitter receptors along a severity gradient•Repeats >1,000 CTGs occur in all individuals; lengths correlate with mis-splicing•GRIP1 mis-splicing perturbs kinesin association and may alter synaptic trafficking•Expression changes suggest neuroinflammation and downregulation of neuronal genes
Otero et al. characterize DM1 frontal cortex transcriptomes and observe extreme CTG repeat lengths even in individuals showing modest mis-splicing. Mis-splicing is enriched in ion channels, neurotransmitter receptors, and synaptic scaffolds, in some cases potentially altering synaptic trafficking. Inferred expression signatures from microglia and endothelial cells suggest neuroinflammation.
Approximately 13% of boys with Duchenne muscular dystrophy (DMD) have a nonsense mutation in the dystrophin gene, resulting in a premature stop codon in the corresponding mRNA and failure to generate ...a functional protein. Ataluren (PTC124) enables ribosomal readthrough of premature stop codons, leading to production of full-length, functional proteins.
This Phase 2a open-label, sequential dose-ranging trial recruited 38 boys with nonsense mutation DMD. The first cohort (n = 6) received ataluren three times per day at morning, midday, and evening doses of 4, 4, and 8 mg/kg; the second cohort (n = 20) was dosed at 10, 10, 20 mg/kg; and the third cohort (n = 12) was dosed at 20, 20, 40 mg/kg. Treatment duration was 28 days. Change in full-length dystrophin expression, as assessed by immunostaining in pre- and post-treatment muscle biopsy specimens, was the primary endpoint.
Twenty three of 38 (61%) subjects demonstrated increases in post-treatment dystrophin expression in a quantitative analysis assessing the ratio of dystrophin/spectrin. A qualitative analysis also showed positive changes in dystrophin expression. Expression was not associated with nonsense mutation type or exon location. Ataluren trough plasma concentrations active in the mdx mouse model were consistently achieved at the mid- and high- dose levels in participants. Ataluren was generally well tolerated.
Ataluren showed activity and safety in this short-term study, supporting evaluation of ataluren 10, 10, 20 mg/kg and 20, 20, 40 mg/kg in a Phase 2b, double-blind, long-term study in nonsense mutation DMD.
ClinicalTrials.gov NCT00264888.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Myotonic dystrophy (DM) is a rare, inherited disorder with multi-systemic effects that impact the skeletal muscles, eyes, heart, skin and gastrointestinal, endocrine, respiratory, and central nervous ...systems. DM is divided into two subtypes: DM1 can present from early childhood through adulthood and also has a congenital form (cDM) while DM2 typically manifests during mid-adulthood. Both forms are progressive with no approved treatments, and unmet need for disease-modifying therapies remains high. This study interrogated health insurance claims data to explore the clinical experience, healthcare resource utilization (HCRU), and all-cause costs for DM.
A total of 8541 patients with DM and 242 patients with cDM and their matched controls were selected from a database of over 200 million claimants. HCRU and all-cause costs, including pharmacy, outpatient, and inpatient services, were analyzed across four years in 12-month follow-up periods. Mean all-cause costs per DM patient were high in each of the four periods (range $14,640-$16,704) and showed a steady increase from 13 to 23 months on, while the control group mean costs declined from $9671 in the first 12 months after the index event, to approach the US population average ($5193) over time. For cDM, the highest mean costs were in the first 12-months ($66,496 vs. $2818 for controls), and remained high (above $17,944) across all subsequent periods, while control mean costs approached $0. For DM and cDM, HCRU was higher compared to controls across all study periods and all-cause healthcare costs were mostly driven by inpatient and outpatient encounters. Analysis of all diagnosis codes over the study period (comorbidities) demonstrated an elevated comorbidity profile consistent with the clinical profile of DM.
This study is among the first to utilize claims data to increase understanding of the clinical experience and health economic outcomes associated with DM. The markedly elevated HCRU patterns and comorbidity profile presented here add to the broad body of scientific and clinical knowledge on DM. These insights can inform clinical care and support the development of disease modifying and/or symptom-targeting therapies that address the multi-systemic, progressive nature of DM.
Introduction/Aims
We studied a patient with a congenital myasthenic syndrome (CMS) caused by a dominant mutation in the synaptotagmin 2 gene (SYT2) and compared the clinical features of this patient ...with those of a previously described patient with a recessive mutation in the same gene.
Methods
We performed electrodiagnostic (EDX) studies, genetic studies, muscle biopsy, microelectrode recordings and electron microscopy (EM).
Results
Both patients presented with muscle weakness and bulbar deficits, which were worse in the recessive form. EDX studies showed presynaptic failure, which was more prominent in the recessive form. Microelectrode studies in the dominant form showed a marked reduction of the quantal content, which increased linearly with higher frequencies of nerve stimulation. The MEPP frequencies were normal at rest but increased markedly with higher frequencies of nerve stimulation. The EM demonstrated overdeveloped postsynaptic folding, and abundant endosomes, multivesicular bodies and degenerative lamellar bodies inside small nerve terminals.
Discussion
The recessive form of CMS caused by a SYT2 mutation showed far more severe clinical manifestations than the dominant form. The pathogenesis of the dominant form likely involves a dominant‐negative effect due to disruption of the dual function of synaptotagmin as a Ca2+‐sensor and modulator of synaptic vesicle exocytosis.
A. Pes cavus in a patient with a heterozygous mutation in the CB2 domain of synaptotagmin 2 (SYT2). B. Schematic view of SYT2 showing the identified mutation. C. Repetitive stimulation demonstrating presynaptic failure of neuromuscular transmission. D. Distorted neuromuscular junction showing intermingled extensions of the nerve terminal (black asterisks) and Schwann cell (white asterisks), endosomes (white arrowheads), coated pits (black arrows), and a lamellar body (LB). Markers are: 5 mV and 5 ms in C, and 0.6 μm in D.
Characterization of the pluripotent “ground state” has led to a greater understanding of species‐specific stem cell differences and has imparted an appreciation of the pluripotency continuum that ...exists in stem cells in vitro. Pluripotent stem cells are functionally coupled via connexins that serve in gap junctional intercellular communication (GJIC) and here we report that the level of connexin expression in pluripotent stem cells depends upon the state in which stem cells exist in vitro. Human and mouse pluripotent stem cells stabilized in a developmentally primitive or “naïve” state exhibit significantly less connexin expression compared with stem cells which are “primed” for differentiation. This dynamic connexin expression pattern may be governed, in part, by differential regulation by pluripotency transcription factors expressed in each cell state. Species‐specific differences do exist, however, with mouse stem cells expressing several additional connexin transcripts not found in human pluripotent stem cells. Moreover, pharmacological inhibition of GJIC shows limited impact on naïve human stem cell survival, self‐renewal, and pluripotency but plays a more significant role in primed human pluripotent stem cells. However, CRISPR‐Cas9 gene ablation of Cx43 in human and mouse primed and naïve pluripotent stem cells reveals that Cx43 is dispensable in each of these four pluripotent stem cell types.
Schematic model depicting expression levels of connexins relative to the pluripotency state in which human and mouse stem cells exist in vitro. Stem cells existing in the naïve ground state express lower overall connexin levels but are still capable of gap junctional intercellular communication. As pluripotent stem cells become “primed” for differentiation, connexin expression (most notably Cx43) is significantly upregulated. Importantly, human and mouse stem cells existing at both ends of the pluripotency continuum appear to retain their signature pluripotency profiles, even in the absence of Cx43.
Abstract
Myotonic dystrophy type 1 is a dominantly inherited multisystemic disease caused by CTG tandem repeat expansions in the DMPK 3′ untranslated region. These expanded repeats are transcribed ...and produce toxic CUG RNAs that sequester and inhibit activities of the MBNL family of developmental RNA processing factors. Although myotonic dystrophy is classified as a muscular dystrophy, the brain is also severely affected by an unusual cohort of symptoms, including hypersomnia, executive dysfunction, as well as early onsets of tau/MAPT pathology and cerebral atrophy. To address the molecular and cellular events that lead to these pathological outcomes, we recently generated a mouse Dmpk CTG expansion knock-in model and identified choroid plexus epithelial cells as particularly affected by the expression of toxic CUG expansion RNAs.
To determine if toxic CUG RNAs perturb choroid plexus functions, alternative splicing analysis was performed on lateral and hindbrain choroid plexi from Dmpk CTG knock-in mice. Choroid plexus transcriptome-wide changes were evaluated in Mbnl2 knockout mice, a developmental-onset model of myotonic dystrophy brain dysfunction. To determine if transcriptome changes also occurred in the human disease, we obtained post-mortem choroid plexus for RNA-seq from neurologically unaffected (two females, three males; ages 50–70 years) and myotonic dystrophy type 1 (one female, three males; ages 50–70 years) donors. To test that choroid plexus transcriptome alterations resulted in altered CSF composition, we obtained CSF via lumbar puncture from patients with myotonic dystrophy type 1 (five females, five males; ages 35–55 years) and non-myotonic dystrophy patients (three females, four males; ages 26–51 years), and western blot and osmolarity analyses were used to test CSF alterations predicted by choroid plexus transcriptome analysis.
We determined that CUG RNA induced toxicity was more robust in the lateral choroid plexus of Dmpk CTG knock-in mice due to comparatively higher Dmpk and lower Mbnl RNA levels. Impaired transitions to adult splicing patterns during choroid plexus development were identified in Mbnl2 knockout mice, including mis-splicing previously found in Dmpk CTG knock-in mice. Whole transcriptome analysis of myotonic dystrophy type 1 choroid plexus revealed disease-associated RNA expression and mis-splicing events. Based on these RNA changes, predicted alterations in ion homeostasis, secretory output and CSF composition were confirmed by analysis of myotonic dystrophy type 1 CSF. Our results implicate choroid plexus spliceopathy and concomitant alterations in CSF homeostasis as an unappreciated contributor to myotonic dystrophy type 1 CNS pathogenesis.
Myotonic dystrophy type 1 is a neuromuscular disease that also severely affects the brain. Using mouse models and patient samples, Nutter et al. describe how this disease alters the gene expression program of cells that produce CSF and propose that these alterations are linked to disease-associated CNS symptoms.
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 β TGFβ-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 TGFβ 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.
Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Through genetic mapping of disease ...loci and whole-exome sequencing in four unrelated multiplex families presenting with severe AMC, we identified biallelic loss-of-function mutations in LGI4 (leucine-rich glioma-inactivated 4). LGI4 is a ligand secreted by Schwann cells that regulates peripheral nerve myelination via its cognate receptor ADAM22 expressed by neurons. Immunolabeling experiments and transmission electron microscopy of the sciatic nerve from one of the affected individuals revealed a lack of myelin. Functional tests using affected individual-derived iPSCs showed that these germline mutations caused aberrant splicing of the endogenous LGI4 transcript and in a cell-based assay impaired the secretion of truncated LGI4 protein. This is consistent with previous studies reporting arthrogryposis in Lgi4-deficient mice due to peripheral hypomyelination. This study adds to the recent reports implicating defective axoglial function as a key cause of AMC.
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