CTG repeat dynamics and DMPK methylation in MSH2 depleted myotonic dystrophy type 1 human pluripotent stem cells

Myotonic dystrophy type 1 is caused by an expanded, unstable CTG repeat in the 3’UTR region of the DMPK gene. The mismatch repair pathway, especially MSH2, has been linked to this unstable character as evidenced in MSH2 knock–out mouse models and MSH2 knock-down human cell models. In addition, the methylation status of the region flanking the repeat is also involved in this instability. However, the effect of MSH2 knock out on repeat size, repeat instability and methylation upstream of the CTG repeat is still unclear. Therefore, we explored a possible correlation between repeat size or instability and methylation by using human pluripotent stem cell models (hPSC) carrying the DM1 mutation with a knock-out of MSH2 obtained by CRISPR/Cas9 genome editing. Repeat instability was measured by PacBio sequencing of PCR fragments spanning the repeat, allowing an accurate assessment of the repeat length. DNA methylation data was obtained by bisulphite treated massive parallel sequencing. Our data shows that in MSH2-/- cells, the region upstream of the CTG repeat loses its methylation gradually over time and observed CTG repeat stabilization. In MSH2+/+ control lines, the repeat continues to expand and DNA remains methylated. We hypothesize that there may be an association between repeat size and methylation upstream of the CTG repeat, as MSH2-/- results in halting CTG expansion, as well as upstream demethylation. Our MSH2-/- hPSC are therefore an excellent model to assess mechanisms of instability, making it possible to develop in vitro methods that eventually will lead to a reduced somatic mosaicism in patients, resulting in an older age of onset and less severe symptoms. We anticipate that the knowledge gained has the potential to be extrapolated to other trinucleotide repeat diseases (TNR). In conclusion, we expect that this analysis will be a major step towards improving understanding of the causes and mechanisms of repeat instability in multiple TNR diseases.


Silvie Franck(1)
Lise Barbé(2)
Yannick de Vlaeminck(3)
Simon Ardui(4)
Claudia Spits(1)
Dominika Dziedzicka(1)
Pierre Hilven(1),
Stella Lanni(5)
Christopher Pearson(5)
Joris R. Vermeesch(4)
Karen Sermon(1)


Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Brussels, 1090, Belgium(1)
Department of Neurological disease, Gladstone Institute, San Francisco, California, United States(2)
Laboratory of Molecular and Cellular Therapy, Vrije Universiteit Brussel, Jette, 1090, Belgium(3)
Center of Human Genetics, University Hospital Leuven, KU Leuven, Leuven, 3000, Belgium(4)
Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Ontario, M5G 0A4, Canada(5)

Presenting author

Silvie Franck, PhD researcher, Vrije Universiteit Brussel
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