Splicing biomarkers of disease severity in myotonic dystrophy

Nakamori, Mikihito; Sobczak, Krzysztof; Puwanant, Araya; Welle, Steve; Eichinger, Katy; Pandya, Shree; Dekdebrun, Jeannne; Heatwole, Chad; McDermott, Michael P.; Chen, Tian; Cline, Melissa; Tawil, Rabi; Osborne, Robert J.; Wheeler, Thurman M.; Swanson, Maurice S.; Moxley, Richard T.; Thornton, Charles A.

doi:10.1002/ana.23992

Cited by 216 publications

(333 citation statements)

References 47 publications

(145 reference statements)

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“…1D). The ΔΨ range in this patient cohort is expected, as myopathic severity (Nakamori et al 2013) and MBNL loss of function (Wagner et al 2016) are also highly variable. Indeed, rank ordering of the adult muscle data sets according to inferred MBNL concentration ([MBNL] inferred ), an approximation of MBNL levels in patient muscle based on a set of 46 validated AS events (Wagner et al 2016), revealed a correlation between functional MBNL level and abnormal MEF2D α 2 exon exclusion (Fig.…”

Section: Severe Rna Misprocessing In Cdmmentioning

confidence: 82%

Disrupted prenatal RNA processing and myogenesis in congenital myotonic dystrophy

et al. 2017

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Myotonic dystrophy type 1 (DM1) is a CTG microsatellite expansion (CTG exp ) disorder caused by expression of CUG exp RNAs. These mutant RNAs alter the activities of RNA processing factors, including MBNL proteins, leading to re-expression of fetal isoforms in adult tissues and DM1 pathology. While this pathogenesis model accounts for adult-onset disease, the molecular basis of congenital DM (CDM) is unknown. Here, we test the hypothesis that disruption of developmentally regulated RNA alternative processing pathways contributes to CDM disease. We identify prominent alternative splicing and polyadenylation abnormalities in infant CDM muscle, and, although most are also misregulated in adult-onset DM1, dysregulation is significantly more severe in CDM. Furthermore, analysis of alternative splicing during human myogenesis reveals that CDM-relevant exons undergo prenatal RNA isoform transitions and are predicted to be disrupted by CUG exp -associated mechanisms in utero. To test this possibility and the contribution of MBNLs to CDM pathogenesis, we generated mouse Mbnl double (Mbnl1; Mbnl2) and triple (Mbnl1; Mbnl2; Mbnl3) muscle-specific knockout models that recapitulate the congenital myopathy, gene expression, and spliceopathy defects characteristic of CDM. This study demonstrates that RNA misprocessing is a major pathogenic factor in CDM and provides novel mouse models to further examine roles for cotranscriptional/post-transcriptional gene regulation during development.

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Section: Severe Rna Misprocessing In Cdmmentioning

confidence: 82%

Disrupted prenatal RNA processing and myogenesis in congenital myotonic dystrophy

et al. 2017

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“…RNAs harboring a long stretch of repeats are thought to fold into stable structures and sequester RNA binding proteins, which, in turn, sets off a molecular cascade leading to neurodegeneration (7). In myotonic dystrophy, sequestration and functional disruption of the muscleblind-like family of RNA binding proteins are associated with specific splicing and expression changes in affected tissues (5,(8)(9)(10)(11)(12).Sequestration of one or more RNA binding proteins into pathological RNA foci has also been proposed in ALS/FTD linked to C9orf72 expansion (1, 13-16). It is anticipated, but has not been demonstrated, that sequestration of RNA binding proteins into expanded GGGGCC RNA foci may lead to major RNA processing alterations as in myotonic dystrophy.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration

Lagier‐Tourenne

Baughn

Rigo

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

521

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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.E xpanded hexanucleotide repeats in the first intron of the chromosome 9 open reading frame 72 (C9orf72) gene were recently identified (1, 2) as the most common genetic cause of ALS, frontotemporal degeneration (FTD), or concomitant ALS/ FTD (3, 4). The mechanisms by which the expanded repeats cause neurodegeneration are unknown, but leading candidate mechanisms are RNA-mediated toxicity, loss of the C9orf72 gene function (from reduced C9orf72 produced by the allele with the expansion), or a combination of the two.RNA-mediated toxicity from nucleotide repeat expansion was initially described for CUG expansion in the RNA encoded by the DMPK gene in myotonic muscular dystrophy (5). A consensus view is that RNA toxicity plays a crucial role in a variety of repeat expansion disorders (6). A hallmark of these disorders is the accumulation of expanded transcripts into nuclear RNA foci (7). RNAs harboring a long stretch of repeats are thought to fold into stable structures and sequester RNA binding proteins, which, in turn, sets off a molecular cascade leading to neurodegeneration (7). In myotonic dystrophy, sequestration and functional disruption of the muscleblind-like family of RNA binding proteins are associated with specific splicing and expression changes in affected tissues (5,(8)(9)(10)(11)(12).Sequestration of one or more RNA binding proteins into pathological RNA foci has ...

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“…Their adverse effects are mediated through sequestration of various proteins including the muscleblind-like (MBNL) family of splicing factors. These proteins are functionally depleted by the expanded repeats in DM1 and DM2 which leads to abnormalities in many pathways of RNA metabolism including alternative splicing, a molecular hallmark of DM [4][5][6][7][8] . In DM adult skeletal muscle abnormal expression of embryonic splicing isoforms has been reported for a few hundred genes 4 .…”

Section: Rnamentioning

confidence: 99%

“…These proteins are functionally depleted by the expanded repeats in DM1 and DM2 which leads to abnormalities in many pathways of RNA metabolism including alternative splicing, a molecular hallmark of DM [4][5][6][7][8] . In DM adult skeletal muscle abnormal expression of embryonic splicing isoforms has been reported for a few hundred genes 4 . Given the importance of alternative splicing as one of the biomarkers of disease severity and therapeutic response, accurate quantification of splicing variants could facilitate their refinement for clinical applications.…”

Section: Rnamentioning

confidence: 99%

Quantitative methods to monitor RNA biomarkers in myotonic dystrophy

Wojciechowska¹,

Sobczak²,

Sedehizadeh³

et al. 2018

Neuromuscular Disorders

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Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are human neuromuscular disorders associated with mutations of simple repetitive sequences in affected genes. The abnormal expansion of CTG repeats in the 3′-UTR of the DMPK gene elicits DM1, whereas elongated CCTG repeats in intron 1 of ZNF9/CNBP triggers DM2. Pathogenesis of both disorders is manifested by nuclear retention of expanded repeatcontaining RNAs and aberrant alternative splicing. The precise determination of absolute numbers of mutant RNA molecules is important for a better understanding of disease complexity and for accurate evaluation of the efficacy of therapeutic drugs. We present two quantitative methods, Multiplex Ligation-Dependent Probe Amplification and droplet digital PCR, for studying the mutant DMPK transcript (DMPK exp RNA) and the aberrant alternative splicing in DM1 and DM2 human tissues and cells. We demonstrate that in DM1, the DMPK exp RNA is detected in higher copy number than its normal counterpart. Moreover, the absolute number of the mutant transcript indicates its low abundance with only a few copies per cell in DM1 fibroblasts. Most importantly, in conjunction with fluorescence in-situ hybridization experiments, our results suggest that in DM1 fibroblasts, the vast majority of nuclear RNA foci consist of a few molecules of DMPK exp RNA.Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are adult onset muscular dystrophies leading to disability and shortened lifespan. Both belong to a larger group of human disorders associated with mutational expansions of simple repetitive sequences within specific genes. The mutation that causes DM1 results from the expansion of CTG repeats in the 3′-UTR of the DMPK gene; whereas DM2 is associated with intronic elongation of CCTG repeats in ZNF9/CNBP gene 1,2 . Pathogenesis of both diseases is mediated by a toxic RNA gain-of-function mechanism manifested by nuclear retention of expanded CUG-and CCUG-harboring RNAs which aggregate within nucleoprotein foci 3 . Their adverse effects are mediated through sequestration of various proteins including the muscleblind-like (MBNL) family of splicing factors. These proteins are functionally depleted by the expanded repeats in DM1 and DM2 which leads to abnormalities in many pathways of RNA metabolism including alternative splicing, a molecular hallmark of DM [4][5][6][7][8] . In DM adult skeletal muscle abnormal expression of embryonic splicing isoforms has been reported for a few hundred genes 4 . Given the importance of alternative splicing as one of the biomarkers of disease severity and therapeutic response, accurate quantification of splicing variants could facilitate their refinement for clinical applications. Specific oligonucleotide-based microarrays and next-generation sequencing have been used for profiling and identification of many aberrantly spliced transcripts. However, their routine application for precise quantification remains limited due to low quantitative parameters of these methods, restriction to highly expressed genes, high costs a...

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Splicing biomarkers of disease severity in myotonic dystrophy

Cited by 216 publications

References 47 publications

Disrupted prenatal RNA processing and myogenesis in congenital myotonic dystrophy

Disrupted prenatal RNA processing and myogenesis in congenital myotonic dystrophy

Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration

Quantitative methods to monitor RNA biomarkers in myotonic dystrophy

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