DM1 repeat instability in human gametes, embryos and embryonic stem cells

Scriptie/masterproef: Doctoral Thesis


Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder with as primary symptoms myotonia, muscular dystrophy, cataracts, cardiac conductance disturbance and cognitive impairment. The molecular basis of the disorder lies in the instability of a CTG triplet repeat in the 3' UTR of the DM1 protein kinase gene (DMPK). The severity of the disease and the age of onset vary with the number of repeats: in normal individuals, the repeat size ranges from 3 to 37 repeats, whereas patients with DM1 exhibit expansions of >50 repeats and, often, as many as several thousand repeats in the congenital form of the disease.
Expanded CTG repeats are unstable and usually increase in number in successive generations. Also in somatic cells, a high level of mosaicism can be detected, depending on the patient's age and repeat number and on the tissues examined. More and more data become available that lead us to unravel the molecular pathogenesis of this disease. But questions remain on the timing of the highly unstable intergenerational transmissions. Currently, the working hypothesis is that the instability may occur at different stages of DNA interactions, such as DNA replication before meiosis, double-strand break formation during meiosis, and DNA repair after meiosis. Little information is available, about the stability of trinucleotide repeats in human early embryos and in female gametes that can confirm these hypotheses.

At our center, preimplantation genetic diagnosis (PGD) for DM1 has been done since 1995. This has given us the unique opportunity to study female gametes and preimplantation embryos shown to be affected after PGD and donated for research by the patients, thus enabling us to close the gap left by the analysis of blood and offspring. These studies showed that enlarged DM1 CTG repeats exists in (im)mature oocytes which is evidence for a prezygotic event during female gametogenesis. Additionally, it was published that enlarged CAG repeats were detected in spermatogonia of HD patients, even in spermatogonia that had not completed the first meiotic divisions, which is indicating that repeat instability in male gametogenesis is not meiosis-dependent. In DM1 preimplantation embryos, from zygotes to blastocysts no mosaicism was detected also suggesting that the intergen-erational repeat instability occurs during gametogenesis.
Since 1998, early human preimplantation embryos can be cultured in vitro as human embryonic stem cells (HESC). ES cells are derived from the ICM cells of blastocysts and have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into all three embryonic germ layers and trophoblast. Because of these characteristics, they are of significant interest as a renewable source of cell populations for different applications.
An important field of research on HESC focuses on the study of the mechanisms of cell differentiation and developmental biology. Another major research area on HESC concentrates on their potentially promising use in cell replacement therapies for the treatment of degenerative human diseases. Finally, because animal models are not fully representative and experiments on humans are restricted, derivation of HESC lines known to be carriers of a monogenic disease can offer the opportunity of an in vitro model of the disease. Those affected HESC lines would be a readily accessible source for pharmacogenetic tests or in vitro gene therapy experiments.
Datum Prijs24 apr 2009
Toekennende instantie
  • Vrije Universiteit Brussel
BegeleiderKaren Sermon (Promotor), Ingeborg Liebaers (Co-promotor), Jacques De Grève (Jury), Mark Van De Casteele (Jury), Leo van Grunsven (Jury), Christopher Pearson (Jury), Gert Matthijs (Jury), B. Loeys (Jury) & Frank Kooy (Jury)

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