CTG Repeat Expansion Research Articles

A CTG repeat-selective chemical screen identifies microtubule inhibitors as selective modulators of toxic CUG RNA levels

A CTG repeat expansion in the DMPK gene is the causative mutation of myotonic dystrophy type 1 (DM1). Transcription of the expanded CTG repeat produces toxic gain-of-function CUG RNA, leading to disease symptoms. A screening platform that targets production or stability of the toxic CUG RNA in a selective manner has the potential to provide new biological and therapeutic insights. A DM1 HeLa cell model was generated that stably expresses a toxic r(CUG)480 and an analogous r(CUG)0 control from DMPK and was used to measure the ratio-metric level of r(CUG)480 versus r(CUG)0. This DM1 HeLa model recapitulates pathogenic hallmarks of DM1, including CUG ribonuclear foci and missplicing of pre-mRNA targets of the muscleblind (MBNL) alternative splicing factors. Repeat-selective screening using this cell line led to the unexpected identification of multiple microtubule inhibitors as hits that selectively reduce r(CUG)480 levels and partially rescue MBNL-dependent missplicing. These results were validated by using the Food and Drug Administration-approved clinical microtubule inhibitor colchicine in DM1 mouse and primary patient cell models. The mechanism of action was found to involve selective reduced transcription of the CTG expansion that we hypothesize to involve the LINC (linker of nucleoskeleton and cytoskeleton) complex. The unanticipated identification of microtubule inhibitors as selective modulators of toxic CUG RNA opens research directions for this form of muscular dystrophy and may shed light on the biology of CTG repeat expansion and inform therapeutic avenues. This approach has the potential to identify modulators of expanded repeat-containing gene expression for over 30 microsatellite expansion disorders.

O.32Genome editing of expanded CTG repeats within the human DMPK gene reduces nuclear RNA foci in muscle of DM1 mice

O.32Genome editing of expanded CTG repeats within the human DMPK gene reduces nuclear RNA foci in muscle of DM1 mice

RNA sequencing reveals abnormal LDB3 splicing in sudden cardiac death

The aim of this study is to determine the molecular mechanism of sudden death in a previously healthy patient. Clinical exome sequencing revealed I536T-RBM20 variant, which alters RNA splicing of TTN and is causative for dilated cardiomyopathy. Comprehensive RNA sequencing (RNA-seq) was also performed in the patient samples and the control samples. Splicing abnormality was compared in cardiac muscle and skeletal muscle. RNA-seq analysis of the cardiac and skeletal muscle showed abnormal splicing of LDB3, not of TTN. Exon 11 of LDB3 was abnormally included in the patient samples compared with the control samples. This abnormal LDB3 splicing pattern in skeletal muscle has been reported in myotonic dystrophy type 1 (DM1) patients. We, thus, confirmed that the patient had expanded CTG repeat in DMPK and the diagnosis was genetically DM1. This finding suggest that one of the molecular mechanisms of sudden cardiac death in this asymptomatic subclinical DM1 patient might be LDB3 abnormal splicing due to the CTG repeat in DMPK, rather than RBM20 variant. RNA-seq analysis is useful to determine the exact molecular diagnosis for sudden cardiac death.

MSH3 modifies somatic instability and disease severity in Huntington's and myotonic dystrophy type 1.

The mismatch repair gene MSH3 has been implicated as a genetic modifier of the CAG·CTG repeat expansion disorders Huntington's disease and myotonic dystrophy type 1. A recent Huntington's disease genome-wide association study found rs557874766, an imputed single nucleotide polymorphism located within a polymorphic 9 bp tandem repeat in MSH3/DHFR, as the variant most significantly associated with progression in Huntington's disease. Using Illumina sequencing in Huntington's disease and myotonic dystrophy type 1 subjects, we show that rs557874766 is an alignment artefact, the minor allele for which corresponds to a three-repeat allele in MSH3 exon 1 that is associated with a reduced rate of somatic CAG·CTG expansion (P = 0.004) and delayed disease onset (P = 0.003) in both Huntington's disease and myotonic dystrophy type 1, and slower progression (P = 3.86 × 10-7) in Huntington's disease. RNA-Seq of whole blood in the Huntington's disease subjects found that repeat variants are associated with MSH3 and DHFR expression. A transcriptome-wide association study in the Huntington's disease cohort found increased MSH3 and DHFR expression are associated with disease progression. These results suggest that variation in the MSH3 exon 1 repeat region influences somatic expansion and disease phenotype in Huntington's disease and myotonic dystrophy type 1, and suggests a common DNA repair mechanism operates in both repeat expansion diseases.

Expansion of trinucleotide CTG repeats in the TCF4 gene as a marker of fuchs’ endothelial corneal dystrophy

Fuchs endothelial corneal dystrophy (FECD) is an inherited severe and progressive disease, characterized by endothelial cell density decrease and increasing corneal edema. FECD development may be linked to expanded trinucleotide repeat, CTG, in the third intron of the TCF4 gene. The study focuses on estimating the prevalence of expanded CTG repeat in TCF4 gene in the Russian population, in patients with normal cornea and in patients with FECD (by applying triplet repeat PCR technique and capillary electrophoresis). 51 patients with FECD and 38 patients with normal cornea were examined. The estimation of the number of CTG triplet repeats in TCF4 gene determination is the veracious laboratory marker of FECD.

Genome Editing of Expanded CTG Repeats within the Human DMPK Gene Reduces Nuclear RNA Foci in the Muscle of DM1 Mice.

Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion located in the 3′ UTR of the DMPK gene. Expanded DMPK transcripts aggregate into nuclear foci and alter the function of RNA-binding proteins, leading to defects in the alternative splicing of numerous pre-mRNAs. To date, there is no curative treatment for DM1. Here we investigated a gene-editing strategy using the CRISPR-Cas9 system from Staphylococcus aureus (Sa) to delete the CTG repeats in the human DMPK locus. Co-expression of SaCas9 and selected pairs of single-guide RNAs (sgRNAs) in cultured DM1 patient-derived muscle line cells carrying 2,600 CTG repeats resulted in targeted DNA deletion, ribonucleoprotein foci disappearance, and correction of splicing abnormalities in various transcripts. Furthermore, a single intramuscular injection of recombinant AAV vectors expressing CRISPR-SaCas9 components in the tibialis anterior muscle of DMSXL (myotonic dystrophy mouse line carrying the human DMPK gene with >1,000 CTG repeats) mice decreased the number of pathological RNA foci in myonuclei. These results establish the proof of concept that genome editing of a large trinucleotide expansion is feasible in muscle and may represent a useful strategy to be further developed for the treatment of myotonic dystrophy.

Dysregulation of Circular RNAs in Myotonic Dystrophy Type 1.

Circular RNAs (circRNAs) constitute a recently re-discovered class of non-coding RNAs functioning as sponges for miRNAs and proteins, affecting RNA splicing and regulating transcription. CircRNAs are generated by “back-splicing”, which is the linking covalently of 3′- and 5′-ends of exons. Thus, circRNA levels might be deregulated in conditions associated with altered RNA-splicing. Significantly, growing evidence indicates their role in human diseases. Specifically, myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG repeats in the DMPK gene which results in abnormal mRNA-splicing. In this investigation, circRNAs expressed in DM1 skeletal muscles were identified by analyzing RNA-sequencing data-sets followed by qPCR validation. In muscle biopsies, out of nine tested, four transcripts showed an increased circular fraction: CDYL, HIPK3, RTN4_03, and ZNF609. Their circular fraction values correlated with skeletal muscle strength and with splicing biomarkers of disease severity, and displayed higher values in more severely affected patients. Moreover, Receiver-Operating-Characteristics curves of these four circRNAs discriminated DM1 patients from controls. The identified circRNAs were also detectable in peripheral-blood-mononuclear-cells (PBMCs) and the plasma of DM1 patients, but they were not regulated significantly. Finally, increased circular fractions of RTN4_03 and ZNF609 were also observed in differentiated myogenic cell lines derived from DM1 patients. In conclusion, this pilot study identified circRNA dysregulation in DM1 patients.

Precise small-molecule cleavage of an r(CUG) repeat expansion in a myotonic dystrophy mouse model.

Myotonic dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by an expanded CTG repeat that is transcribed into r(CUG)exp The RNA repeat expansion sequesters regulatory proteins such as Muscleblind-like protein 1 (MBNL1), which causes pre-mRNA splicing defects. The disease-causing r(CUG)exp has been targeted by antisense oligonucleotides, CRISPR-based approaches, and RNA-targeting small molecules. Herein, we describe a designer small molecule, Cugamycin, that recognizes the structure of r(CUG)exp and cleaves it in both DM1 patient-derived myotubes and a DM1 mouse model, leaving short repeats of r(CUG) untouched. In contrast, oligonucleotides that recognize r(CUG) sequence rather than structure cleave both long and short r(CUG)-containing transcripts. Transcriptomic, histological, and phenotypic studies demonstrate that Cugamycin broadly and specifically relieves DM1-associated defects in vivo without detectable off-targets. Thus, small molecules that bind and cleave RNA have utility as lead chemical probes and medicines and can selectively target disease-causing RNA structures to broadly improve defects in preclinical animal models.

Abnormal nuclear aggregation and myotube degeneration in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is caused by CTG nucleotide repeat expansions in the 3'-untranslated region (3'-UTR) of the dystrophia myotonica protein kinase (DMPK) gene. The expanded CTG repeats encode toxic CUG RNAs that cause disease, largely through RNA gain-of-function. DM1 is a fatal disease characterized by progressive muscle wasting, which has no cure. Regenerative medicine has emerged as a promising therapeutic modality for DM1, especially with the advancement of induced pluripotent stem (iPS) cell technology and therapeutic genome editing. However, there is an unmet need to identify in vitro outcome measures to demonstrate the therapeutic effects prior to in vivo clinical trials. In this study, we examined the muscle regeneration (myotube formation) in normal and DM1 myoblasts in vitro to establish outcome measures for therapeutic monitoring. We found normal proliferation of DM1 myoblasts, but abnormal nuclear aggregation during the early stage myotube formation, as well as myotube degeneration during the late stage of myotube formation. We concluded that early abnormal nuclear aggregation and late myotube degeneration offer easy and sensitive outcome measures to monitor therapeutic effects in vitro.

Expanded CUG Repeats Trigger Disease Phenotype and Expression Changes through the RNAi Machinery in C. elegans

Myotonic dystrophy type 1 is an autosomal-dominant inherited disorder caused by the expansion of CTG repeats in the 3′ untranslated region of the DMPK gene. The RNAs bearing these expanded repeats have a range of toxic effects. Here we provide evidence from a Caenorhabditis elegans myotonic dystrophy type 1 model that the RNA interference (RNAi) machinery plays a key role in causing RNA toxicity and disease phenotypes. We show that the expanded repeats systematically affect a range of endogenous genes bearing short non-pathogenic repeats and that this mechanism is dependent on the small RNA pathway. Conversely, by perturbating the RNA interference machinery, we reversed the RNA toxicity effect and reduced the disease pathogenesis. Our results unveil a role for RNA repeats as templates (based on sequence homology) for moderate but constant gene silencing. Such a silencing effect affects the cell steady state over time, with diverse impacts depending on tissue, developmental stage, and the type of repeat. Importantly, such a mechanism may be common among repeats and similar in human cells with different expanded repeat diseases.

Nuclear Envelope Transmembrane Proteins in Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder with predominant myotonia and muscular dystrophy which is caused by CTG-repeat expansions in the DMPK gene. These repeat expansions are transcribed and the resulting mRNA accumulates RNA-binding proteins involved in splicing, resulting in a general splicing defect. We observed nuclear envelope (NE) alterations in DM1 primary myoblasts. These included invaginations of the NE as well as an altered composition of the nuclear lamina. Specifically, we investigated NE transmembrane proteins (NETs) in DM1 primary myoblasts, staining to determine if their distribution was altered compared to controls and if this could contribute to these structural defects. We also tested the expression of these NETs in muscle and how localization changes in the DM1 primary myoblasts undergoing differentiation in vitro to myotubes. We found no changes in the localization of the tested NETs, but most tended to exhibit reduced expression with increasing DMPK-repeat length. Nonetheless, the DM1 patient expression range was within the expression range of the controls. Additionally, we found a down-regulation of the possible nesprin 1 giant isoform in DM1 primary myoblasts which could contribute to the increased NE invaginations. Thus, nesprin 1 may be an interesting target for further investigation in DM1 disease pathology.

Therapeutic Genome Editing for Myotonic Dystrophy Type 1Using CRISPR/Cas9.

Myotonic dystrophy type 1 (DM1) is caused by a CTG nucleotide repeat expansion within the 3′ UTR of the Dystrophia Myotonica protein kinase gene. In this study, we explored therapeutic genome editing using CRISPR/Cas9 via targeted deletion of expanded CTG repeats and targeted insertion of polyadenylation signals in the 3′ UTR upstream of the CTG repeats to eliminate toxic RNA CUG repeats. We found paired SpCas9 or SaCas9 guide RNA induced deletion of expanded CTG repeats. However, this approach incurred frequent inversion in both the mutant and normal alleles. In contrast, the insertion of polyadenylation signals in the 3′ UTR upstream of the CTG repeats eliminated toxic RNA CUG repeats, which led to phenotype reversal in differentiated neural stem cells, forebrain neurons, cardiomyocytes, and skeletal muscle myofibers. We concluded that targeted insertion of polyadenylation signals in the 3′ UTR is a viable approach to develop therapeutic genome editing for DM1.

Generation of induced pluripotent stem cell line(ZZUi006-A)from a patient with myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is a dominantly inherited neurodegenerative disease caused by a CTG repeat expansion close to the DMPK gene. The fibroblasts from a patient with DM1 were successfully converted to induced pluripotent stem cells (iPSCs), designated ZZUi006-A, by employing episomal plasmids expressing OCT3/4, SOX2, KLF4, LIN28, L-MYC. The ZZUi006-A cell line may provide a robust platform for further study of DM1 pathogenesis as well as drug testing and gene therapy research.

Abstract 520: Overexpression of a Non-muscle RBFOX2 Splice Isoform Induce Cardiac Arrhythmias in Myotonic Dystrophy

Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disease caused by a CTG repeat expansion in 3’-UTR of DMPK gene. DM1 affects multiple tissues, but cardiac dysfunctions are the second leading cause of death which often include conduction defects and arrhythmias. The best characterized pathogenic mechanism of DM1 is toxic gain-of-function of expanded CUG repeat (CUG exp ) RNA that accumulates to form ribonuclear foci causing sequestration of MBNL and overexpression of CELF1 family of splicing factors. However, loss of MBNL or gain of CELF1 activity does not explain the cardiac phenotypes observed in DM1. Here we report that steady-state protein levels of RBFOX2, a critical splicing regulator, are drastically upregulated in DM1 heart tissue. This is accompanied by aberrant skipping of a muscle-specific 43bp exon in RBFOX2 transcript, resulting in selective up-regulation of the non-muscle RBFOX2 splice isoform in adult cardiomyocytes. We demonstrate that expression of CUG exp RNA in DM1 affects RBFOX2 in two distinct ways: reduced expression of a subset of microRNAs causes de-repression of RBFOX2 protein production; and CELF1 overexpression promotes skipping of the muscle-specific exon of RBFOX2 . Remarkably, tet -inducible overexpression of the non-muscle RBFOX2 isoform, or CRISPR/ Cas9 mediated deletion of the muscle-specific RBFOX2 exon in the mouse heart results in prolonged PR and QRS intervals, slower conduction velocity and spontaneous cardiac arrhythmias that mirror human DM1 pathology. Integration of RBFOX2-CLIP with RNA-seq data from cardiomyocytes of mice expressing the non-muscle RBFOX2 isoform identified a core network of mRNA splicing defects in genes encoding sodium, potassium, and calcium ion channels and key excitation-contraction coupling proteins that are similarly misspliced in hearts of DM1 patients. Thus, we have uncovered a central role for RBFOX2 isoform switching in DM1 cardiac pathogenesis and identified the molecular mechanisms that may alter electrophysiological properties and thereby induce cardiac arrhythmias in the DM1 heart.

Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells.

CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3′-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.

Incidence of amplification failure in DMPK allele due to allelic dropout event in a diagnostic laboratory

BackgroundMyotonic dystrophy type 1 (DM1) is caused by an expanded CTG repeat in the non-coding 3’ UTR of the DMPK gene. PCR and Southern Blot Analysis (SBA) of long-range PCR represent the routine molecular testing most widely used for DM1 diagnosis.However, in these conventional methods artifacts such as allele dropout (ADO) represent a risk of misdiagnosis for DM1. Subjects, who show a single product by conventional methods, require a complementary technique such as triplet repeat primed PCR (TP-PCR). ObjectTo estimate and minimize the incidence of allele dropout event in our diagnostic molecular laboratory by the use of new kit TP-PCR-based. MethodsWe retrospectively studied 190 DMPK alleles, on blood samples from to ninety-five subjects, previously genotyped by traditional methods to validate a new assay. The pedigree of a DM1 family was used to expand our analysis. ResultsTP-PCR assay correctly identified all 95/95 (100%) subjects and these results were in agreement with the other molecular laboratory. By conventional methods the amplification failure due to allele dropout in our cohort was in 12/190 (6.3%) DMPK alleles analyzed. When these 12 alleles were detected and solved by new assay, we found that the 2.6% was caused by primer sequence-dependent and the remaining 3.6% by polymerase-hindering secondary structures. ConclusionsAllele dropout could be considered as a potentially important problem in DM1 diagnosis that may lead to the attribution of a wrong genotype with long-term consequences for both proband and family.

Unusual association of a unique CAG interruption in 5' of DM1 CTG repeats with intergenerational contractions and low somatic mosaicism.

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder associated with high variability of symptoms and anticipation. DM1 is caused by an unstable CTG repeat expansion that usually increases in successive generations and tissues. DM1 family pedigrees have shown that ∼90% and 10% of transmissions result in expansions and contractions of the CTG repeat, respectively. To date, the mechanisms of CTG repeat contraction remain poorly documented in DM1. In this report, we identified two new DM1 families with apparent contractions and no worsening of DM1 symptoms in two and three successive maternal transmissions. A new and unique CAG interruption was found in 5' of the CTG expansion in one family, whereas multiple 5' CCG interruptions were detected in the second family. We showed that these interruptions are associated with maternal intergenerational contractions and low somatic mosaicism in blood. By specific triplet-prime PCR, we observed that CTG repeat changes (contractions/expansions) occur preferentially in 3' of the interruptions for both families.

Small Molecules Which Improve Pathogenesis of Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults for which there is currently no treatment. The pathogenesis of this autosomal dominant disorder is associated with the expansion of CTG repeats in the 3′-UTR of the DMPK gene. DMPK transcripts with expanded CUG repeats (CUGexpDMPK) are retained in the nucleus forming multiple discrete foci, and their presence triggers a cascade of toxic events. Thus far, most research emphasis has been on interactions of CUGexpDMPK with the muscleblind-like (MBNL) family of splicing factors. These proteins are sequestered by the expanded CUG repeats of DMPK RNA leading to their functional depletion. As a consequence, abnormalities in many pathways of RNA metabolism, including alternative splicing, are detected in DM1. To date, in vitro and in vivo efforts to develop therapeutic strategies for DM1 have mostly been focused on targeting CUGexpDMPK via reducing their expression and/or preventing interactions with MBNL1. Antisense oligonucleotides targeted to the CUG repeats in the DMPK transcripts are of particular interest due to their potential capacity to discriminate between mutant and normal transcripts. However, a growing number of reports describe alternative strategies using small molecule chemicals acting independently of a direct interaction with CUGexpDMPK. In this review, we summarize current knowledge about these chemicals and we describe the beneficial effects they caused in different DM1 experimental models. We also present potential mechanisms of action of these compounds and pathways they affect which could be considered for future therapeutic interventions in DM1.

A CRISPR‐Based Selective Gene Inhibition Method Reveals Dynamic Features of a Cell Nucleus Nanobody Related to the Disease Myotonic Dystrophy

AbstractA major form of the severe pediatric muscle disease myotonic dystrophy is caused by a CTG repeat expansion in the gene (dmpk) for the enzyme DM protein kinase 1. As a consequence, this mutant allele produces aberrant transcripts that have elongated tracts of CUG repeats. These RNAs appear to stall within the cell nucleus, congressing in small, discrete bodies situated away from their site of gene transcription. Here, clustered regularly interspaced short palindromic repeats (CRISPR)‐based approach is deployed that reveals that these nuclear nanobodies are not static structures. When the transcription of the dmpk gene is halted by a targeted CRISPR‐based inhibition method, the preexisting transcripts delocalize from the nuclear bodies, showing that they are continuously trafficking through and are not stalled there in an accretion mode. This report is an example of how a novel method can address a nanoscale dimension of molecular cell biology in the nucleus, one with direct medical relevance.

Mechanisms of skeletal muscle wasting in a mouse model for myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disease resulting in severe muscle weakening and wasting. DM1 is caused by expansion of CTG repeats in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. We have developed an inducible, skeletal muscle-specific mouse model of DM1 (CUG960) that expresses 960 CUG repeat-expressing animals (CUG960) in the context of human DMPK exons 11-15. CUG960 RNA-expressing mice induced at postnatal day 1, as well as adult-onset animals, show clear, measurable muscle wasting accompanied by severe histological defects including central myonuclei, reduced fiber cross-sectional area, increased percentage of oxidative myofibers, the presence of nuclear RNA foci that colocalize with Mbnl1 protein, and increased Celf1 protein in severely affected muscles. Importantly, muscle loss, histological abnormalities and RNA foci are reversible, demonstrating recovery upon removal of toxic RNA. RNA-seq and protein array analysis indicate that the balance between anabolic and catabolic pathways that normally regulate muscle mass may be disrupted by deregulation of platelet derived growth factor receptor β signaling and the PI3K/AKT pathways, along with prolonged activation of AMP-activated protein kinase α signaling. Similar changes were detected in DM1 skeletal muscle compared with unaffected controls. The mouse model presented in this paper shows progressive skeletal muscle wasting and has been used to identify potential molecular mechanisms underlying skeletal muscle loss. The reversibility of the phenotype establishes a baseline response for testing therapeutic approaches.