Hit-and-run silencing of endogenous DUX4 by targeting DNA hypomethylation on D4Z4 repeats in facioscapulohumeral muscular dystrophy
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE201185 | GEO | 2024/03/31
REPOSITORIES: GEO
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Project description:Facioscapulohumeral muscular dystrophy (FSHD), a progressive skeletal muscle disorder, is epigenetically characterized with DNA hypomethylation of D4Z4 repeats in 4q35 region, allowing aberrant DUX4 expression. Sustainable DUX4 suppression is a promising therapeutic clue to prevent disease progression, but most of the supposed methods depend on expression of their mediator biochemical entity, potentially narrowing QoL of individuals with FSHD in the clinical context. In this study, we report that by applying hit-and-run silencing with dCas9-mediated epigenetic editing targeting DNA hypomethylation on D4Z4 repeats, we could achieve suppression of endogenous DUX4 in our FSHD patients-derived iPSC model. Notably, DNA methylation was significantly upregulated in FSHD cells and suppression effect was observed at least two weeks after intervention, which was not the case by transient treatment of typical dCas9-KRAB alone. Off-target analysis showed that despite the potential genome-wide risk in DNA methylation, the impact on transcriptome was limited. We propose that hit-and-run silencing can be a promising option to prevent disease progression with minimum intervention for individuals with FSHD, motivating further study for clinical development.
2024-03-31 | GSE199690 | GEO
Project description:Facioscapulohumeral muscular dystrophy (FSHD), a progressive skeletal muscle disorder, is epigenetically characterized with DNA hypomethylation of D4Z4 repeats in 4q35 region, allowing aberrant DUX4 expression. Sustainable DUX4 suppression is a promising therapeutic clue to prevent disease progression, but most of the supposed methods depend on expression of their mediator biochemical entity, potentially narrowing QoL of individuals with FSHD in the clinical context. In this study, we report that by applying hit-and-run silencing with dCas9-mediated epigenetic editing targeting DNA hypomethylation on D4Z4 repeats, we could achieve suppression of endogenous DUX4 in our FSHD patients-derived iPSC model. Notably, DNA methylation was significantly upregulated in FSHD cells and suppression effect was observed at least two weeks after intervention, which was not the case by transient treatment of typical dCas9-KRAB alone. Off-target analysis showed that despite the potential genome-wide risk in DNA methylation, the impact on transcriptome was limited. We propose that hit-and-run silencing can be a promising option to prevent disease progression with minimum intervention for individuals with FSHD, motivating further study for clinical development.
2024-03-31 | GSE201178 | GEO
Project description:Hit-and-run silencing of endogenous DUX4 by targeting DNA hypomethylation on D4Z4 repeats in facioscapulohumeral muscular dystrophy
| PRJNA828606 | ENA
Project description:Hit-and-run silencing of endogenous DUX4 by targeting DNA hypomethylation on D4Z4 repeats in facioscapulohumeral muscular dystrophy [RNA-seq]
| PRJNA828600 | ENA
Project description:Hit-and-run silencing of endogenous DUX4 by targeting DNA hypomethylation on D4Z4 repeats in facioscapulohumeral muscular dystrophy [EPIC Methylation]
| PRJNA821190 | ENA
Project description:Background and objectivesThe objective of this study was to examine whether the regional methylation levels at the most distal D4Z4 repeat units (RU) in the 4qA-permissive haplotype were associated with disease severity and progression in facioscapulohumeral muscular dystrophy type 1 (FSHD1).MethodsThis 21-year, retrospective, observational cohort study was conducted at the Fujian Neuromedical Center (FNMC) in China. Methylation levels of the most distal D4Z4 RU, including 10 CpGs, were assessed in all participants by bisulfite sequencing. Patients with FSHD1 were stratified into 4 groups based on methylation percentage quartiles, including LM1 (low methylation), LM2 (low to intermediate methylation), LM3 (intermediate to high methylation), and highest methylation (HM) levels. Patients received evaluations of motor function focusing on lower extremity (LE) progression at baseline and in follow-ups. FSHD clinical score (CS), age-corrected clinical severity scale (ACSS), and modified Rankin scale were used to assess motor function.ResultsThe methylation levels of the 10 CpGs were significantly lower in all 823 patients with genetically confirmed FSHD1 than in 341 healthy controls (HCs). CpG6 methylation levels could distinguish the following: (1) patients with FSHD1 from HCs; (2) symptomatic from asymptomatic/unaffected patients; (3) patients with LE involvement from those without LE involvement, with AUCs (95% CI) of 0.9684 (0.9584-0.9785), 0.7417 (0.6903-0.7931), and 0.6386 (0.5816-0.6956), respectively. Lower CpG6 methylation levels were correlated with higher CS (r = -0.392), higher ACSS (r = -0.432), and earlier onset age of first-ever muscle weakness (r = 0.297). For the LM1, LM2, LM3, and HM groups, the respective proportions of LE involvement were 52.9%, 44.2%, 36.9%, and 23.4%; and onset ages of LE involvement were 20, 26.5, 25, and 26.5 years. Cox regression analysis-adjusted for sex, age at examination, D4Z4 RU, and 4qA/B haplotype-showed that the LM1, LM2, and LM3 groups (i.e., groups with lower methylation levels) had a higher risk of independent ambulation loss, with HRs (95% CI) of 3.523 (1.565-7.930), 3.356 (1.458-7.727), and 2.956 (1.245-7.020), respectively.Discussion4q35 distal D4Z4 hypomethylation is correlated with disease severity and progression to lower extremity involvement.
| S-EPMC10382269 | biostudies-literature
Project description:PurposeFacioscapulohumeral muscular dystrophy (FSHD) is a common adult muscular dystrophy. Over 95% of FSHD cases are associated with contraction of the D4Z4 tandem repeat (~3.3 kb per unit) at 4q35 with a specific genomic configuration (haplotype) called 4qA. Molecular diagnosis of FSHD typically requires pulsed-field gel electrophoresis with Southern blotting. We aim to develop novel genomic and computational methods for characterising D4Z4 repeat numbers in FSHD.MethodsWe leveraged a single-molecule optical mapping platform that maps locations of restriction enzyme sites on high molecular weight (>150 kb) DNA molecules. We developed bioinformatics methods to address several challenges, including the differentiation of 4qA with 4qB alleles, the differentiation of 4q35 and 10q26 segmental duplications, the quantification of repeat numbers with different enzymes that may or may not have recognition sites within D4Z4 repeats. We evaluated the method on 25 human subjects (13 patients, 3 individual control subjects, 9 control subjects from 3 families) labelled by the Nb.BssSI and/or Nt.BspQI enzymes.ResultsWe demonstrated that the method gave a direct quantitative measurement of repeat numbers on D4Z4 repeats with 4qA allelic configuration and the levels of postzygotic mosaicism. Our method had high concordance with Southern blots from several cohorts on two platforms (Bionano Saphyr and Bionano Irys), but with improved quantification of repeat numbers.ConclusionWhile the study is limited by small sample size, our results demonstrated that single-molecule optical mapping is a viable approach for more refined analysis on genotype-phenotype relationships in FSHD, especially when postzygotic mosaicism is present.
| S-EPMC7029236 | biostudies-literature
Project description:Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) has an unusual pathogenic mechanism. FSHD is caused by deletion of a subset of D4Z4 macrosatellite repeat units in the subtelomere of chromosome 4q. Recent studies provide compelling evidence that a retrotransposed gene in the D4Z4 repeat, DUX4, is expressed in the human germline and then epigenetically silenced in somatic tissues. In FSHD, the combination of inefficient chromatin silencing of the D4Z4 repeat and polymorphisms on the FSHD-permissive alleles that stabilize the DUX4 mRNAs emanating from the repeat result in inappropriate DUX4 protein expression in muscle cells. FSHD is thereby the first example of a human disease caused by the inefficient repression of a retrogene in a macrosatellite repeat array.
| S-EPMC3092836 | biostudies-literature
Project description:Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy characterized by an asymmetric progressive weakness and wasting of the facial, shoulder and upper arm muscles, frequently accompanied by hearing loss and retinal vasculopathy. FSHD is an autosomal dominant disease linked to chromosome 4q35, but the causative gene remains controversial. DUX4 is a leading candidate gene as causative of FSHD. However, DUX4 expression is extremely low in FSHD muscle, and there is no DUX4 animal model that mirrors the pathology in human FSHD. Here, we show that the misexpression of very low levels of human DUX4 in zebrafish development recapitulates the phenotypes seen in human FSHD patients. Microinjection of small amounts of human full-length DUX4 (DUX4-fl) mRNA into fertilized zebrafish eggs caused asymmetric abnormalities such as less pigmentation of the eyes, altered morphology of ears, developmental abnormality of fin muscle, disorganization of facial musculature and/or degeneration of trunk muscle later in development. Moreover, DUX4-fl expression caused aberrant localization of myogenic cells marked with ?-actin promoter-driven enhanced green fluorescent protein outside somite boundary, especially in head region. These abnormalities were rescued by coinjection of the short form of DUX4 (DUX4-s). Our results suggest that the misexpression of DUX4-fl, even at extremely low level, can recapitulate the phenotype observed in FSHD patients in a vertebrate model. These results strongly support the current hypothesis for a role of DUX4 in FSHD pathogenesis. We also propose that DUX4 expression during development is important for the pathogenesis of FSHD.
| S-EPMC3606007 | biostudies-other
Project description:Purpose of reviewFacioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder, which is caused by incomplete repression of the transcription factor double homeobox 4 (DUX4) in skeletal muscle. To date, there is no DUX4-targeting treatment to prevent or delay disease progression. In the present review, we summarize developments in therapeutic strategies with the focus on inhibiting DUX4 and DUX4 target gene expression.Recent findingsDifferent studies show that DUX4 and its target genes can be repressed with genetic therapies using diverse strategies. Additionally, different small compounds can reduce DUX4 and its target genes in vitro and in vivo.SummaryMost studies that show DUX4 repression by genetic therapies have only been tested in vitro. More efforts should be made to test them in vivo for clinical translation. Several compounds have been shown to prevent DUX4 and target gene expression in vitro and in vivo. However, their efficiency and specificity has not yet been shown. With emerging clinical trials, the clinical benefit from DUX4 repression in FSHD will likely soon become apparent.
| S-EPMC7735392 | biostudies-literature