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Expanded CTG repeats within the DMPK 3' UTR causes severe skeletal muscle wasting in an inducible mouse model for myotonic dystrophy.

ABSTRACT: Severe skeletal muscle wasting is the most debilitating symptom experienced by individuals with myotonic dystrophy type 1 (DM1). We present a DM1 mouse model with inducible and skeletal muscle-specific expression of large tracts of CTG repeats in the context of DMPK exon 15. These mice recapitulate many findings associated with DM1 skeletal muscle, such as CUG RNA foci with Muscleblind-like 1 (MBNL1) protein colocalization, misregulation of developmentally regulated alternative splicing events, myotonia, characteristic histological abnormalities, and increased CUGBP1 protein levels. Importantly, this DM1 mouse model recapitulates severe muscle wasting, which has not been reported in models in which depletion of MBNL1 is the main feature. Using these mice, we discovered previously undescribed alternative splicing events that are responsive to CUGBP1 and not MBNL, and these events were found to be misregulated in individuals with DM1. Our results indicate that increased CUGBP1 protein levels are associated with DMPK-CUG RNA expression, suggesting a role for CUGBP1-specific splicing or cytoplasmic functions in muscle wasting.

SUBMITTER: Orengo JP

PROVIDER: S-EPMC2268190 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Variant repeats within the DMPK CTG expansion protect function in myotonic dystrophy type 1.

Project description:Objective:We tested the hypothesis that variant repeat interruptions (RIs) within the DMPK CTG repeat tract lead to milder symptoms compared with pure repeats (PRs) in myotonic dystrophy type 1 (DM1). Methods:We evaluated motor, neurocognitive, and behavioral outcomes in a group of 6 participants with DM1 with RI compared with a case-matched sample of 12 participants with DM1 with PR and a case-matched sample of 12 unaffected healthy comparison participants (UA). Results:In every measure, the RI participants were intermediate between UA and PR participants. For muscle strength, the RI group was significantly less impaired than the PR group. For measures of Full Scale IQ, depression, and sleepiness, all 3 groups were significantly different from each other with UA > RI > PR in order of impairment. The RI group was different from unaffected, but not significantly different from PR (UA > RI = PR) in apathy and working memory. Finally, in finger tapping and processing speed, RI did not differ from UA comparisons, but PR had significantly lower scores than the UA comparisons (UA = RI > PR). Conclusions:Our results support the notion that patients affected by DM1 with RI demonstrate a milder phenotype with the same pattern of deficits as those with PR indicating a similar disease process.

| S-EPMC7428360 | biostudies-literature

The DMPK gene of severely affected myotonic dystrophy patients is hypermethylated proximal to the largely expanded CTG repeat.

Project description:Using methylation-sensitive restriction enzymes, we characterized the methylation pattern on the 5' side of the CTG repeat in the DMPK gene of normal individuals and of patients affected with myotonic dystrophy, showing expansions of the repetitive sequence. The gene segment analyzed corresponds to the genomic SacI-HindIII fragment carrying exons 11-15. There is constitutive methylation in intron 12 at restriction sites of SacII and HhaI, localized 1,159-1,232 bp upstream of the CTG repeat, whereas most, if not all, of the other sites of SacII, HhaI, and HpaII in this region are unmethylated, in normal individuals and most of the patients. In a number of young and severely affected patients, however, complete methylation of these restriction sites was found in the mutated allele. In most of these patients, the onset of the disease was congenital. Preliminary in vivo footprinting data gave evidence for protein-DNA contact in normal genes at an Sp1 consensus binding site upstream of the CTG repeat and for a significant reduction of this interaction in cells with a hypermethylated DMPK gene.

| S-EPMC1376887 | biostudies-other

High throughput screening for expanded CTG repeats in myotonic dystrophy type 1 using melt curve analysis.

Project description:BackgroundMyotonic dystrophy type 1 (DM1) is caused by CTG repeat expansions in the DMPK gene and is the most common form of muscular dystrophy. Patients can have long delays from onset to diagnosis, since clinical signs and symptoms are often nonspecific and overlapping with other disorders. Clinical genetic testing by Southern blot or triplet-primed PCR (TP-PCR) is technically challenging and cost prohibitive for population surveys.MethodsHere, we present a high throughput, low-cost screening tool for CTG repeat expansions using TP-PCR followed by high resolution melt curve analysis with saturating concentrations of SYBR GreenER dye.ResultsWe determined that multimodal melt profiles from the TP-PCR assay are a proxy for amplicon length stoichiometry. In a screen of 10,097 newborn blood spots, melt profile analysis accurately reflected the tri-modal distribution of common alleles from 5 to 35 CTG repeats, and identified the premutation and full expansion alleles.ConclusionWe demonstrate that robust detection of expanded CTG repeats in a single tube can be achieved from samples derived from specimens with minimal template DNA such as dried blood spots (DBS). This technique is readily adaptable to large-scale testing programs such as population studies and newborn screening programs.

| S-EPMC8123750 | biostudies-literature

<i>In Cis</i> Effect of <i>DMPK</i> Expanded Alleles in Myotonic Dystrophy Type 1 Patients Carrying Variant Repeats at 5' and 3' Ends of the CTG Array.

Project description:Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disease caused by a CTG repeat expansion in the 3'-untranslated region (UTR) of DMPK gene. DM1 alleles containing non-CTG variant repeats (VRs) have been described, with uncertain molecular and clinical consequences. The expanded trinucleotide array is flanked by two CpG islands, and the presence of VRs could confer an additional level of epigenetic variability. This study aims to investigate the association between VR-containing DMPK alleles, parental inheritance and methylation pattern of the DM1 locus. The DM1 mutation has been characterized in 20 patients using a combination of SR-PCR, TP-PCR, modified TP-PCR and LR-PCR. Non-CTG motifs have been confirmed by Sanger sequencing. The methylation pattern of the DM1 locus was determined by bisulfite pyrosequencing. We characterized 7 patients with VRs within the CTG tract at 5' end and 13 patients carrying non-CTG sequences at 3' end of the DM1 expansion. DMPK alleles with VRs at 5' end or 3' end were invariably unmethylated upstream of the CTG expansion. Interestingly, DM1 patients with VRs at the 3' end showed higher methylation levels in the downstream island of the CTG repeat tract, preferentially when the disease allele was maternally inherited. Our results suggest a potential correlation between VRs, parental origin of the mutation and methylation pattern of the DMPK expanded alleles. A differential CpG methylation status could play a role in the phenotypic variability of DM1 patients, representing a potentially useful diagnostic tool.

| S-EPMC10299395 | biostudies-literature

Muscular myopathies other than myotonic dystrophy also associated with (CTG)n expansion at the DMPK locus.

Project description:OBJECTIVE:Assess triplet repeat expansion (CTG)n at the 'dystrophia-myotonica protein kinase' (DMPK) locus in muscular myopathies to elucidate its role in myopathic symptoms and enable genetic counseling and prenatal diagnosis in families. METHODS AND RESULTS:Individuals with symptoms of myopathy, hypotonia and controls selected randomly from the population were evaluated for triplet repeat expansion of (CTG)n repeats in the 3'untranslated region (UTR) of DMPK gene, the causative mutation in myotonic dystrophy (DM). DNA was isolated from peripheral blood of 40 individuals; they presented symptoms of muscle myopathy (n = 11), muscle hypotonia (n = 4), members of their families (n = 5) and control individuals from random population (n = 20). Molecular analysis of genomic DNA by polymerase chain reaction (PCR) using primers specific for the DMPK gene encompassing the triplet repeat expansion, showed that all controls (n = 20) gave a 2.1 kb band indicating normal triplet repeat number. Three out of 11 cases (two clinically diagnosed DM and one muscular dystrophy) had an expansion of the (CTG)n repeat in the range of 1000-2100 repeats corresponding to the repeat number in cases of severe DM. Other two of these 11 cases, showed a mild expansion of ~ 66 repeats. Three samples, which included two cases of hypotonia and the father of a subject with muscular dystrophy, also gave a similar repeat expansion (~66 repeats). CONCLUSION:Results suggest a role of (CTG)n expansion at the DMPK locus in unexplained hypotonias and muscular myopathies other than DM. This calls for screening of the triplet repeat expansion at the DMPK locus in cases of idiopathic myopathies and hypotonia.

| S-EPMC3611902 | biostudies-literature

Allele length of the DMPK CTG repeat is a predictor of progressive myotonic dystrophy type 1 phenotypes.

Project description:Myotonic dystrophy type 1 (DM1) is an autosomal dominant inherited disorder caused by expansion of a germline and somatically unstable CTG repeat in the DMPK gene. Previously, CTG repeat length at birth has been correlated to patient age at symptom onset. Attempts to correlate CTG repeat length with progressive DM1 phenotypes, such as muscle power, have proven difficult. To better correlate genotype with progressive phenotypes, we have measured CTG repeat tract length and screened for interrupting variant repeats in 192 study participants from a well-characterized Canadian cohort. We have assessed genotype-phenotype correlations with nine progressive measures of skeletal muscle power and respiratory function. We have built statistical models that include confounding factors such as sex, age, height and weight to further explain variation in muscle power. Our analysis reveals a strong correlation between DM1 genotype and respiratory function and skeletal muscle power, as part of a complex model that includes additional modulators such as sex, age, height, weight and the presence or absence of interrupting variant repeats. Distal skeletal muscle measurements, such as hand pinch and grip strength, show the strongest correlation with disease genotype. Detailed analysis of CTG repeat length, and incorporation of confounding factors, greatly improves the predictive ability of these models. They reveal a greater genetic influence on individual progressive phenotypes than on age at symptom onset and for clinical trials will help optimize stratification and explain patient variability. They will also help practitioners prioritize assessment of the muscular power measurements that correlate best with disease severity.

| S-EPMC6586140 | biostudies-literature

Brain Structural Features of Myotonic Dystrophy Type 1 and their Relationship with CTG Repeats.

Project description:BackgroundFew adequately-powered studies have systematically evaluated brain morphology in adult-onset myotonic dystrophy type 1 (DM1).ObjectiveThe goal of the present study was to determine structural brain differences between individuals with and without adult-onset DM1 in a multi-site, case-controlled cohort. We also explored correlations between brain structure and CTG repeat length.MethodsNeuroimaging data was acquired in 58 unaffected individuals (29 women) and 79 individuals with DM1 (50 women). CTG repeat length, expressed as estimated progenitor allele length (ePAL), was determined by small pool PCR. Statistical models were adjusted for age, sex, site, and intracranial volume (ICV).ResultsICV was reduced in DM1 subjects compared with controls. Accounting for the difference in ICV, the DM1 group exhibited smaller volume in frontal grey and white matter, parietal grey matter as well as smaller volume of the corpus callosum, thalamus, putamen, and accumbens. In contrast, volumes of the hippocampus and amygdala were significantly larger in DM1. Greater ePAL was associated with lower volumes of the putamen, occipital grey matter, and thalamus. A positive ePAL association was observed for amygdala volume and cerebellar white matter.ConclusionsSmaller ICV may be a marker of aberrant neurodevelopment in adult-onset DM1. Volumetric analysis revealed morphological differences, some associated with CTG repeat length, in structures with plausible links to key DM1 symptoms including cognitive deficits and excessive daytime somnolence. These data offer further insights into the basis of CNS disease in DM1, and highlight avenues for further work to identify therapeutic targets and imaging biomarkers.

| S-EPMC7480174 | biostudies-literature

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.

| S-EPMC3000673 | biostudies-literature

Epigenetics of the myotonic dystrophy-associated DMPK gene neighborhood.

Project description:Identify epigenetic marks in the vicinity of DMPK (linked to myotonic dystrophy, DM1) that help explain tissue-specific differences in its expression.At DMPK and its flanking genes (DMWD, SIX5, BHMG1 and RSPH6A), we analyzed many epigenetic and transcription profiles from myoblasts, myotubes, skeletal muscle, heart and 30 nonmuscle samples.In the DMPK gene neighborhood, muscle-associated DNA hypermethylation and hypomethylation, enhancer chromatin, and CTCF binding were seen. Myogenic DMPK hypermethylation correlated with high expression and decreased alternative promoter usage. Testis/sperm hypomethylation of BHMG1 and RSPH6A was associated with testis-specific expression. G-quadruplex (G4) motifs and sperm-specific hypomethylation were found near the DM1-linked CTG repeats within DMPK.Tissue-specific epigenetic features in DMPK and neighboring genes help regulate its expression. G4 motifs in DMPK DNA and RNA might contribute to DM1 pathology.

| S-EPMC4863877 | biostudies-other

Stable Longitudinal Methylation Levels at the CpG Sites Flanking the CTG Repeat of DMPK in Patients with Myotonic Dystrophy Type 1.

Project description:Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystem disorder mainly characterized by gradual muscle loss, weakness, and delayed relaxation after muscle contraction. It is caused by an expanded CTG repeat in the 3' UTR of DMPK, which is transcribed into a toxic gain-of-function mRNA that affects the splicing of a range of other genes. The repeat is unstable, with a bias towards expansions both in somatic cells and in the germline, which results in a tendency for earlier onset with each generation, as longer repeat lengths generally correlate with earlier onset. Previous studies have found hypermethylation in the regions flanking the repeat in congenital onset DM1 and in some patients with non-congenital DM1. We used pyrosequencing to investigate blood methylation levels in 68 patients with non-congenital DM1, compare the methylation levels between the blood and muscle, and assess whether methylation levels change over time in the blood. We found higher methylation levels in the blood of DM1 patients than in healthy controls and especially in the patients who had inherited the disease allele maternally. The methylation levels remained relatively stable over time and are a strong biomarker of the disease, as well as of the maternal inheritance of the disease.

| S-EPMC7465187 | biostudies-literature

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