Myotonic dystrophy type 1 embryonic stem cells show decreased myogenic potential, increased CpG methylation at the DMPK locus and RNA mis-splicing

ABSTRACT Skeletal muscle tissue is severely affected in myotonic dystrophy type 1 (DM1) patients, characterised by muscle weakness, myotonia and muscle immaturity in the most severe congenital form of the disease. Previously, it was not known at what stage during myogenesis the DM1 phenotype appears. In this study we differentiated healthy and DM1 human embryonic stem cells to myoblasts and myotubes and compared their differentiation potential using a comprehensive multi-omics approach. We found myogenesis in DM1 cells to be abnormal with altered myotube generation compared to healthy cells. We did not find differentially expressed genes between DM1 and non-DM1 cell lines within the same developmental stage. However, during differentiation we observed an aberrant inflammatory response and increased CpG methylation upstream of the CTG repeat at the myoblast level and RNA mis-splicing at the myotube stage. We show that early myogenesis modelled in hESC reiterates the early developmental manifestation of DM1.

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MSH2 knock-down shows CTG repeat stability and concomitant upstream demethylation at the DMPK locus in myotonic dystrophy type 1 human embryonic stem cells

Human Molecular Genetics ◽

10.1093/hmg/ddaa250 ◽

2020 ◽

Author(s):

Silvie Franck ◽

Lise Barbé ◽

Simon Ardui ◽

Yannick De Vlaeminck ◽

Joke Allemeersch ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Myotonic Dystrophy ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Cpg Methylation ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type ◽

Ctg Repeat

Abstract Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DMPK gene, where expansion size and somatic mosaicism correlates with disease severity and age of onset. While it is known that the mismatch repair protein MSH2 contributes to the unstable nature of the repeat, its role on other disease-related features, such as CpG methylation upstream of the repeat, is unknown. In this study, we investigated the effect of an MSH2 knock-down (MSH2KD) on both CTG repeat dynamics and CpG methylation pattern in human embryonic stem cells (hESC) carrying the DM1 mutation. Repeat size in MSH2 wild type (MSH2WT) and MSH2KD DM1 hESC was determined by PacBio sequencing and CpG methylation by bisulfite massive parallel sequencing. We found stabilization of the CTG repeat concurrent with a gradual loss of methylation upstream of the repeat in MSH2KD cells, while the repeat continued to expand and upstream methylation remained unchanged in MSH2WT control lines. Repeat instability was re-established and biased towards expansions upon MSH2 transgenic re-expression in MSH2KD lines while upstream methylation was not consistently re-established. We hypothesize that the hypermethylation at the mutant DM1 locus is promoted by the MMR machinery and sustained by a constant DNA repair response, establishing a potential mechanistic link between CTG repeat instability and upstream CpG methylation. Our work represents a first step towards understanding how epigenetic alterations and repair pathways connect and contribute to the DM1 pathology.

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MSH2 knock-down shows CTG repeat stability and concomitant upstream demethylation at the DMPK locus in myotonic dystrophy type 1 human embryonic stem cells

10.1101/2020.09.25.313197 ◽

2020 ◽

Author(s):

Silvie Franck ◽

Lise Barbé ◽

Simon Ardui ◽

Yannick De Vlaeminck ◽

Joke Allemeersch ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Myotonic Dystrophy ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Cpg Methylation ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type ◽

Ctg Repeat

AbstractMyotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DMPK gene, where expansion size and somatic mosaicism correlates with disease severity and age of onset. While it is known that the mismatch repair protein MSH2 contributes to the unstable nature of the repeat, its role on other disease-related features, such as CpG methylation upstream of the repeat, is unknown. In this study, we investigated the effect of an MSH2 knock-down (MSH2KD) on both CTG repeat dynamics and CpG methylation pattern in human embryonic stem cells (hESC) carrying the DM1 mutation. Repeat size in MSH2 wild type (MSH2WT) and MSH2KD DM1 hESC was determined by PacBio sequencing and CpG methylation by bisulfite massive parallel sequencing. We found stabilization of the CTG repeat concurrent with a gradual loss of methylation upstream of the repeat in MSH2KD cells, while the repeat continued to expand and upstream methylation remained unchanged in MSH2WT control lines. Repeat instability was re-established and biased towards expansions upon MSH2 transgenic re-expression in MSH2KD lines while upstream methylation was not consistently re-established. We hypothesize that the hypermethylation at the mutant DM1 locus is promoted by the MMR machinery and sustained by a constant DNA repair response, establishing a potential mechanistic link between CTG repeat instability and upstream CpG methylation. Our work represents a first step towards understanding how epigenetic alterations and repair pathways connect and contribute to the DM1 pathology.

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Deletion of the CTG Expansion in Myotonic Dystrophy Type 1 Reverses DMPK Aberrant Methylation in Human Embryonic Stem Cells but not Affected Myoblasts

10.1101/631457 ◽

2019 ◽

Cited By ~ 2

Author(s):

Shira Yanovsky-Dagan ◽

Ester Bnaya ◽

Manar Abu Diab ◽

Tayma Handal ◽

Fouad Zahdeh ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Myotonic Dystrophy ◽

Embryonic Stem ◽

Aberrant Methylation ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type ◽

Gene Correction ◽

Large Expansion

ABSTRACTMyotonic dystrophy type 1 (DM1) results from a CTG repeat expansion in the 3’-UTR of DMPK. When the repeat extensively expands, this results in DMPK aberrant methylation, reduction in SIX5 transcription and the development of the congenital form of the disease. To explore whether hypermethylation could be reversed in DM1 embryonic stem cells (hESCs) and patient myoblasts, we monitored methylation levels following removal of the expanded repeat by CRISPR/Cas9-mediated editing. Excision of the repeat in undifferentiated hESCs (CTG2000) resets the locus by abolishing abnormal methylation and H3K9me3 enrichment, and rescues SIX5 transcription. In contrast, in affected myoblasts methylation levels remain unchanged following deletion of a large expansion (CTG2600). Altogether, this provides evidence for a transition from a reversible to an irreversible heterochromatin state by the DM1 mutation upon cell differentiation. These findings should be taken into account when considering gene correction in congenital DM1 and potentially other epigenetically regulated disorders.

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Uncovering the Role of Hypermethylation by CTG Expansion in Myotonic Dystrophy Type 1 Using Mutant Human Embryonic Stem Cells

Stem Cell Reports ◽

10.1016/j.stemcr.2015.06.003 ◽

2015 ◽

Vol 5 (2) ◽

pp. 221-231 ◽

Cited By ~ 20

Author(s):

Shira Yanovsky-Dagan ◽

Michal Avitzour ◽

Gheona Altarescu ◽

Paul Renbaum ◽

Talia Eldar-Geva ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Myotonic Dystrophy ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type ◽

Ctg Expansion

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Dosage effect of multiple genes accounts for multisystem disorder of myotonic dystrophy type 1

Cell Research ◽

10.1038/s41422-019-0264-2 ◽

2019 ◽

Vol 30 (2) ◽

pp. 133-145 ◽

Cited By ~ 3

Author(s):

Qi Yin ◽

Hongye Wang ◽

Na Li ◽

Yifu Ding ◽

Zhenfei Xie ◽

...

Keyword(s):

Stem Cells ◽

Myotonic Dystrophy ◽

Dosage Reduction ◽

Embryonic Stem ◽

Mutant Mice ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type ◽

Multisystem Disorder ◽

Multiple Genes

AbstractMultisystem manifestations in myotonic dystrophy type 1 (DM1) may be due to dosage reduction in multiple genes induced by aberrant expansion of CTG repeats in DMPK, including DMPK, its neighboring genes (SIX5 or DMWD) and downstream MBNL1. However, direct evidence is lacking. Here, we develop a new strategy to generate mice carrying multigene heterozygous mutations to mimic dosage reduction in one step by injection of haploid embryonic stem cells with mutant Dmpk, Six5 and Mbnl1 into oocytes. The triple heterozygous mutant mice exhibit adult-onset DM1 phenotypes. With the additional mutation in Dmwd, the quadruple heterozygous mutant mice recapitulate many major manifestations in congenital DM1. Moreover, muscle stem cells in both models display reduced stemness, providing a unique model for screening small molecules for treatment of DM1. Our results suggest that the complex symptoms of DM1 result from the reduced dosage of multiple genes.

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Dosage effect of multiple genes accounts for multisystem disorder of myotonic dystrophy type 1

10.1101/658526 ◽

2019 ◽

Author(s):

Qi Yin ◽

Hongye Wang ◽

Zhenfei Xie ◽

Lifang Jin ◽

Yifu Ding ◽

...

Keyword(s):

Stem Cells ◽

Myotonic Dystrophy ◽

Gene Dosage ◽

Embryonic Stem ◽

Mutant Mice ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type ◽

Multisystem Disorder ◽

Multiple Genes

AbstractMultisystem manifestations in myotonic dystrophy type 1 (DM1) may be due to dosage reduction in multiple genes induced by aberrant expansion of CTG repeats in DMPK, including Dmpk and its neighboring genes (Six5 or Dmwd) and downstream Mbnl1. However, the direct evidences are lack. Here, we develop a new strategy to generate mice carrying multigene mutations in one step by injection of haploid embryonic stem cells with mutant Dmpk, Six5 and Mbnl1 into oocytes. The triple heterozygous mutant mice exhibit adult-onset DM1 phenotypes. With the additional mutation in Dmwd, quadruple heterozygous mutant mice recapitulate many major manifestations in congenital DM1. Moreover, muscle stem cells in both models display reduced stemness. Our results suggest that the complex symptoms of DM1 result from the reduced gene dosage of multiple genes.

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Stem cells reveal mechanisms of myotonic dystrophy type 1

Nature Reviews Neurology ◽

10.1038/nrneurol.2011.79 ◽

2011 ◽

Vol 7 (6) ◽

pp. 301-301

Author(s):

Heather Wood

Keyword(s):

Stem Cells ◽

Myotonic Dystrophy ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type

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DMPK hypermethylation in sperm cells of myotonic dystrophy type 1 patients

European Journal of Human Genetics ◽

10.1038/s41431-021-00999-3 ◽

2021 ◽

Author(s):

Shira Yanovsky-Dagan ◽

Eliora Cohen ◽

Pauline Megalli ◽

Gheona Altarescu ◽

Oshrat Schonberger ◽

...

Keyword(s):

Myotonic Dystrophy ◽

Embryonic Stem ◽

Severe Form ◽

Sperm Cells ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type ◽

Viable Sperm ◽

Ctg Expansion ◽

Male Patients

AbstractMyotonic dystrophy type 1 (DM1) is an autosomal dominant muscular dystrophy that results from a CTG expansion (50–4000 copies) in the 3′ UTR of the DMPK gene. The disease is classified into four or five somewhat overlapping forms, which incompletely correlate with expansion size in somatic cells of patients. With rare exception, it is affected mothers who transmit the congenital (CDM1) and most severe form of the disease. Why CDM1 is hardly ever transmitted by fathers remains unknown. One model to explain the almost exclusive transmission of CDM1 by affected mothers suggests a selection against hypermethylated large expansions in the germline of male patients. By assessing DNA methylation upstream to the CTG expansion in motile sperm cells of four DM1 patients, together with availability of human embryonic stem cell (hESCs) lines with paternally inherited hypermethylated expansions, we exclude the possibility that DMPK hypermethylation leads to selection against viable sperm cells (as indicated by motility) in DM1 patients.

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