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Detection of slipped-DNAs at the trinucleotide repeats of the myotonic dystrophy type I disease locus in patient tissues.

ABSTRACT: Slipped-strand DNAs, formed by out-of-register mispairing of repeat units on complementary strands, were proposed over 55 years ago as transient intermediates in repeat length mutations, hypothesized to cause at least 40 neurodegenerative diseases. While slipped-DNAs have been characterized in vitro, evidence of slipped-DNAs at an endogenous locus in biologically relevant tissues, where instability varies widely, is lacking. Here, using an anti-DNA junction antibody and immunoprecipitation, we identify slipped-DNAs at the unstable trinucleotide repeats (CTG)n•(CAG)n of the myotonic dystrophy disease locus in patient brain, heart, muscle and other tissues, where the largest expansions arise in non-mitotic tissues such as cortex and heart, and are smallest in the cerebellum. Slipped-DNAs are shown to be present on the expanded allele and in chromatinized DNA. Slipped-DNAs are present as clusters of slip-outs along a DNA, with each slip-out having 1-100 extrahelical repeats. The allelic levels of slipped-DNA containing molecules were significantly greater in the heart over the cerebellum (relative to genomic equivalents of pre-IP input DNA) of a DM1 individual; an enrichment consistent with increased allelic levels of slipped-DNA structures in tissues having greater levels of CTG instability. Surprisingly, this supports the formation of slipped-DNAs as persistent mutation products of repeat instability, and not merely as transient mutagenic intermediates. These findings further our understanding of the processes of mutation and genetic variation.

SUBMITTER: Axford MM

PROVIDER: S-EPMC3868534 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Slipped-strand DNAs formed by long (CAG)*(CTG) repeats: slipped-out repeats and slip-out junctions.

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Project description: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.

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Expanded CTG repeat demarcates a boundary for abnormal CpG methylation in myotonic dystrophy patient tissues.

Project description:Myotonic dystrophy (DM1) affects multiple organs, shows age-dependent progression and is caused by CTG expansions at the DM1 locus. We determined the DM1 CpG methylation profile and CTG length in tissues from DM1 foetuses, DM1 adults, non-affected individuals and transgenic DM1 mice. Analysis included CTCF binding sites upstream and downstream of the CTG tract, as methylation-sensitive CTCF binding affects chromatinization and transcription of the DM1 locus. In humans, in a given foetus, expansions were largest in heart and smallest in liver, differing by 40-400 repeats; in adults, the largest expansions were in heart and cerebral cortex and smallest in cerebellum, differing by up to 5770 repeats in the same individual. Abnormal methylation was specific to the mutant allele. In DM1 adults, heart, liver and cortex showed high-to-moderate methylation levels, whereas cerebellum, kidney and skeletal muscle were devoid of methylation. Methylation decreased between foetuses and adults. Contrary to previous findings, methylation was not restricted to individuals with congenital DM1. The expanded repeat demarcates an abrupt boundary of methylation. Upstream sequences, including the CTCF site, were methylated, whereas the repeat itself and downstream sequences were not. In DM1 mice, expansion-, tissue- and age-specific methylation patterns were similar but not identical to those in DM1 individuals; notably in mice, methylation was present up- and downstream of the repeat, but greater upstream. Thus, in humans, the CpG-free expanded CTG repeat appears to maintain a highly polarized pattern of CpG methylation at the DM1 locus, which varies markedly with age and tissues.

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RNA interference targeting CUG repeats in a mouse model of myotonic dystrophy.

Project description:Myotonic dystrophy type 1 (DM1) is an RNA dominant disease caused by expression of DM protein kinase (DMPK) transcripts that contain an expanded CUG repeat (CUG(exp)). The toxic mRNA localizes to nuclear foci and sequesters proteins involved in the regulation of alternative splicing, such as, muscleblind-like 1 (MBNL1). Here, we used synthetic short interfering RNAs (siRNAs) to target CUG repeats and test the concept that inhibiting the expression of CUG(exp) RNA can mitigate features of DM1 in transgenic mice. Intramuscular injection and electroporation of siRNA resulted in ~70-80% downregulation of CUG(exp) transcripts. A limited survey of endogenous mouse transcripts that contain nonexpanded CUG or CAG repeats showed that most were not affected, though Txlnb containing (CUG)(9) was significantly reduced. By this strategy, the number and intensity of CUG(exp) nuclear foci were reduced and splicing of MBNL1-dependent exons was improved. These data suggest that the expanded CUG repeats are a potential target for allele-selective RNA interference.

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Molecular and Clinical Implications of Variant Repeats in Myotonic Dystrophy Type 1.

Project description:Myotonic dystrophy type 1 (DM1) is one of the most variable monogenic diseases at phenotypic, genetic, and epigenetic level. The disease is multi-systemic with the age at onset ranging from birth to late age. The underlying mutation is an unstable expansion of CTG repeats in the DMPK gene, varying in size from 50 to >1000 repeats. Generally, large expansions are associated with an earlier age at onset. Additionally, the most severe, congenital DM1 form is typically associated with local DNA methylation. Genetic variability of DM1 mutation is further increased by its structural variations due to presence of other repeats (e.g., CCG, CTC, CAG). These variant repeats or repeat interruptions seem to confer an additional level of epigenetic variability since local DNA methylation is frequently associated with variant CCG repeats independently of the expansion size. The effect of repeat interruptions on DM1 molecular pathogenesis is not investigated enough. Studies on patients indicate their stabilizing effect on DMPK expansions because no congenital cases were described in patients with repeat interruptions, and the age at onset is frequently later than expected. Here, we review the clinical relevance of repeat interruptions in DM1 and genetic and epigenetic characteristics of interrupted DMPK expansions based on patient studies.

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