Splicing Biomarkers of Disease Severity in Myotonic Dystrophy
ABSTRACT To develop RNA splicing biomarkers of disease severity and therapeutic response in myotonic dystrophy type 1 (DM1) and type 2 (DM2).
In a discovery cohort we used microarrays to perform global analysis of alternative splicing in DM1 and DM2. The newly identified splicing changes were combined with previous data to create a panel of 50 putative splicing defects. In a validation cohort of 50 DM1 subjects we measured the strength of ankle dorsiflexion (ADF) and then obtained a needle biopsy of tibialis anterior (TA) to analyze splice events in muscle RNA. The specificity of DM-associated splicing defects was assessed in disease controls. The CTG expansion size in muscle tissue was determined by Southern blot. The reversibility of splicing defects was assessed in transgenic mice by using antisense oligonucleotides (ASOs) to reduce levels of toxic RNA.
Forty-two splicing defects were confirmed in TA muscle in the validation cohort. Among these, 20 events showed graded changes that correlated with ADF weakness. Five other splice events were strongly affected in DM1 subjects with normal ADF strength. Comparison to disease controls and mouse models indicated that splicing changes were DM-specific, mainly attributable to MBNL1 sequestration, and reversible in mice by targeted knockdown of toxic RNA. Splicing defects and weakness were not correlated with CTG expansion size in muscle tissue.
Alternative splicing changes in skeletal muscle may serve as biomarkers of disease severity and therapeutic response in myotonic dystrophy. ANN NEUROL 2013. © 2013 American Neurological Association.
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ABSTRACT: Expanded hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9orf72) gene are the most common genetic cause of ALS and frontotemporal degeneration (FTD). Here, we identify nuclear RNA foci containing the hexanucleotide expansion (GGGGCC) in patient cells, including white blood cells, fibroblasts, glia, and multiple neuronal cell types (spinal motor, cortical, hippocampal, and cerebellar neurons). RNA foci are not present in sporadic ALS, familial ALS/FTD caused by other mutations (SOD1, TDP-43, or tau), Parkinson disease, or nonneurological controls. Antisense oligonucleotides (ASOs) are identified that reduce GGGGCC-containing nuclear foci without altering overall C9orf72 RNA levels. By contrast, siRNAs fail to reduce nuclear RNA foci despite marked reduction in overall C9orf72 RNAs. Sustained ASO-mediated lowering of C9orf72 RNAs throughout the CNS of mice is demonstrated to be well tolerated, producing no behavioral or pathological features characteristic of ALS/FTD and only limited RNA expression alterations. Genome-wide RNA profiling identifies an RNA signature in fibroblasts from patients with C9orf72 expansion. ASOs targeting sense strand repeat-containing RNAs do not correct this signature, a failure that may be explained, at least in part, by discovery of abundant RNA foci with C9orf72 repeats transcribed in the antisense (GGCCCC) direction, which are not affected by sense strand-targeting ASOs. Taken together, these findings support a therapeutic approach by ASO administration to reduce hexanucleotide repeat-containing RNAs and raise the potential importance of targeting expanded RNAs transcribed in both directions.Proceedings of the National Academy of Sciences 10/2013; 110(47). DOI:10.1073/pnas.1318835110 · 9.81 Impact Factor
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ABSTRACT: RNA-binding proteins (RBPs) play pivotal roles in multiple cellular pathways from transcription to RNA turnover by interacting with RNA sequence and/or structural elements to form distinct RNA-protein complexes. Since these complexes are required for the normal regulation of gene expression, mutations that alter RBP functions may result in a cascade of deleterious events that lead to severe disease. Here, we focus on a group of hereditary disorders, the microsatellite expansion diseases, which alter RBP activities and result in abnormal neurological and neuromuscular phenotypes. While many of these diseases are classified as adult-onset disorders, mounting evidence indicates that disruption of normal RNA-protein interaction networks during embryogenesis modifies developmental pathways, which ultimately leads to disease manifestations later in life. Efforts to understand the molecular basis of these disorders has already uncovered novel pathogenic mechanisms, including RNA toxicity and repeat-associated non-ATG (RAN) translation, and current studies suggest that additional surprising insights into cellular regulatory pathways will emerge in the future.Advances in Experimental Medicine and Biology 01/2014; 825:353-88. DOI:10.1007/978-1-4939-1221-6_10 · 2.01 Impact Factor
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ABSTRACT: Myotonic dystrophy (DM) of type 1 and 2 (DM1 and DM2) are inherited autosomal dominant diseases caused by dynamic and unstable expanded microsatellite sequences (CTG and CCTG, respectively) in the non-coding regions of the genes DMPK and ZNF9, respectively. These mutations result in the intranuclear accumulation of mutated transcripts and the mis-splicing of numerous transcripts. This so-called RNA gain of toxic function is the main feature of an emerging group of pathologies known as RNAopathies. Interestingly, in addition to these RNA inclusions, called foci, the presence of neurofibrillary tangles (NFT) in patient brains also distinguishes DM as a tauopathy. Tauopathies are a group of nearly 30 neurodegenerative diseases that are characterized by intraneuronal protein aggregates of the microtubule-associated protein Tau (MAPT) in patient brains. Furthermore, a number of neurodegenerative diseases involve the dysregulation of splicing regulating factors and have been characterized as spliceopathies. Thus, myotonic dystrophies are pathologies resulting from the interplay among RNAopathy, spliceopathy, and tauopathy. This review will describe how these processes contribute to neurodegeneration. We will first focus on the tauopathy associated with DM1, including clinical symptoms, brain histology, and molecular mechanisms. We will also discuss the features of DM1 that are shared by other tauopathies and, consequently, might participate in the development of a tauopathy. Moreover, we will discuss the determinants common to both RNAopathies and spliceopathies that could interfere with tau-related neurodegeneration.Frontiers in Molecular Neuroscience 01/2014; 6:57. DOI:10.3389/fnmol.2013.00057 · 4.08 Impact Factor