CTG repeat instability and myotonic dystrophy
- Mario Gomes-Pereira
- Stéphanie Tomé
- Geneviève Gourdon
- Aline Huguet-Lachon
- Diana Dinca
- Elodie Dandelot
- Sandra Braz
- Aïcha Ali
- Alexis Bertrand
CTG repeat instability and myotonic dystrophy
Myotonic dystrophy type I (DM1) is dominantly inherited, clinically highly variable and is caused by the unstable expansion of a CTG repeat in the 3’UTR of the DMPK on chromosome 19. The normal DMPK gene contains 5-37 CTG repeats in the 3'UTR, while all DM1 patients have repeats expanded from 50 to several thousand CTG trinucleotides. The size of the CTG repeat increases from generation to generation, is generally correlated with clinical severity and age at onset, providing a molecular basis for the anticipation phenomenon observed in DM1 families. Furthermore, the repeat increases with age in several tissues, possibly in relation with the progression of the disease with time. Mutant DMPK mRNAs accumulate in nuclear inclusions, interfering with the activity, localization and/or steady-state levels of RNA-interacting proteins.
These toxic RNA deregulates the splicing program of a subset of developmentally regulated genes in multiple tissues, resulting in a multi-systemic condition. However, recent findings suggest that DM1 molecular pathogenesis is vastly more complex, going beyond spliceopathy, involving changes in gene expression and translation efficiency, antisense transcripts, non-conventional translation and micro-RNA (miRNA) deregulation. A better understanding of the disease pathophysiology is crucial for the rational development of effective therapies targeting the molecular defects that underlie the multi-systemic symptoms that characterize myotonic dystrophy.
Several years ago, we created a transgenic mouse model carrying very large human genomic sequences containing the DMPK gene and the largest CTG repeat introduced in mice so far. These mice show a very high level of CTG repeat instability and reproduce the trans-dominant effect of the mutant DMPK gene.
Our research follows three main axes:
Characterization of the mechanisms involved in trinucleotide repeat instability
Analysis of the CTG repeat length in transgenic mouse tissues and over generations showed that the CTG repeat instability, in our mice, is very similar to the CTG repeat instability observed in DM1 patients. Furthermore it revealed that the CTG surrounding genomic sequences and the human chromatin environment are necessary to recreate the features and the characteristic dynamics of the trinucleotide repeat instability in mice. We have also demonstrated that MMR proteins are the main actor in the formations of CTG expansions. We pursue our studies towards a better characterization of the dynamics of repeat instability. In collaboration with the Necker hospital diagnostic center and the French DM registry DMScope, we have identified very unusual families showing stabilization or contraction of the repeat through successive generations; identification of the mechanisms involved will give new hints for the development of new therapeutics approaches.
Molecular and physiopathological consequences of CTft repeat expansions
The DMPK transgene carrying the expansion is expressed in different mouse tissues and during development, contributing to the development of a variety of symptoms in multiple tissues and organ systems. In the laboratory, we decided to focused on the consequences of the mutation
1) in the central nervous system.
We are using various tools to understand the molecular, cellular and behavioral mechanisms behind the neuropsychological impairment and brain abnormalities observed in DM1 patients.
2) in neonates.
The congenital form of the disease (CDM) is extremely severe at birth and in young children. Using our mouse model showing high mortality in the neonatal period, we intend to identify mechanisms behind very characteristic CDM symptoms such as mental retardation and respiratory failure.
Preclinical gene therapy in DM1 mice
Different groups working on DM1 have dedicated their efforts to the development of gene therapy strategies aiming to destroy the toxic mutant RNA, to correct DM1 splicing defects or to restore the function of the proteins affected by the CUG expansion. Using our transgenic mouse model, we are collaborating with different research groups world-wide in the assessment of therapeutic tools recently developed. With this aim, we have developed various behavioral assays to test different approaches (pharmacological, Antisens oligonucleotides, CRISPR/Cas9, TALEN, AAV).