Demethylation, reactivation, and destabilization of human fragile X full-mutation alleles in mouse embryocarcinoma cells

The major causes of fragile X syndrome are mutational expansion of the CGG repeat in the FMR1 gene, hypermethylation, and transcriptional silencing. M ost fragile X embryos develop somatic mosaicism of disease-causing "full" e xpansions of different lengths. Homogeneity of the mosaic patterns among mu ltiple tissues in the same individual indicates that these previously unsta ble expansions acquire mitotic stability early in fetal life. Since mitotic stability is found strictly associated with hypermethylation in adult tiss ues, current theory has fixed the time of instability to developmental stag es when fully expanded CGG repeats exist in an unmethylated state. We used murine embryocarcinoma (EC) cells (PC13) as a model system of pluripotent e mbryonic cells. Hypermethylated and unmethylated full expansions on human f ragile 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 sta ble in the donors' fibroblasts and in A9 became demethylated, reactivated, and destabilized in undifferentiated EC hybrids. When destabilized expansio ns were reintroduced from EC cells into A9, instability was reversed to sta bility. Our results strongly support the idea that fully expanded alleles a re initially unstable and unmethylated in the human embryo and gain stabili ty upon genetic or epigenetic change of the embryonic cells.