title: Demethylation, reactivation, and destabilization of human fragile X full-mutation alleles in mouse embryocarcinoma cells.
abstract: The major causes of fragile X syndrome are mutational expansion of the CGG repeat in the FMR1 gene, hypermethylation, and transcriptional silencing. Most fragile X embryos develop somatic mosaicism of disease-causing "full" expansions of different lengths. Homogeneity of the mosaic patterns among multiple tissues in the same individual indicates that these previously unstable expansions acquire mitotic stability early in fetal life. Since mitotic stability is found strictly associated with hypermethylation in adult tissues, current theory has fixed the time of instability to developmental stages when fully expanded CGG repeats exist in an unmethylated state. We used murine embryocarcinoma (EC) cells (PC13) as a model system of pluripotent embryonic cells. Hypermethylated and unmethylated full expansions on human fragile X chromosomes were transferred from murine A9 hybrids into EC cells, by means of microcell fusion. As demonstrated in the present study for the first time, even full expansion alleles that were fully methylated and stable in the donors' fibroblasts and in A9 became demethylated, reactivated, and destabilized in undifferentiated EC hybrids. When destabilized expansions were reintroduced from EC cells into A9, instability was reversed to stability. Our results strongly support the idea that fully expanded alleles are initially unstable and unmethylated in the human embryo and gain stability upon genetic or epigenetic change of the embryonic cells.
authors.affiliation: Department of Human Genetics, University Hospital of Ulm, 89073 Ulm, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Wöhrle
authors.forename: U
authors.initials: U
authors.lastname: Salat
authors.forename: H
authors.initials: H
authors.lastname: Hameister
authors.forename: W
authors.initials: W
authors.lastname: Vogel
authors.forename: P
authors.initials: P
authors.lastname: Steinbach
mesh_terms: D000483:Alleles; D000818:Animals; D019175:DNA Methylation; D054278:Embryonal Carcinoma Stem Cells; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000235:genetics*; D018014:Gene Transfer Techniques; D006801:Humans; D051379:Mice; D008938:Mitosis; D008969:Molecular Sequence Data; D009154:Mutation; D014411:Neoplastic Stem Cells / Q000473:pathology; D009419:Nerve Tissue Proteins / Q000235:genetics*; D016601:RNA-Binding Proteins; D014407:Tumor Cells, Cultured
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; C497683:FMR1 protein, human; C497684:Fmr1 protein, mouse