Fully expanded FMR1 CGG repeats exhibit a length- and differentiation-dependent instability in cell hybrids that is independent of DNA methylation

The fragile X syndrome is characterized at the molecular level by expansion and methylation of a CGG trinucleotide repeat located within the FMR1 locu s. The tissues of most full mutation carriers are mosaic for repeat size, b ut these mutational patterns tend to be well conserved when comparing multi ple tissues within an individual, Moreover, full mutation alleles are stabl e in cultured fibroblasts. These observations have been used to suggest tha t fragile X CGG repeat instability normally is limited to a period during e arly embryogenesis, DNA methylation of the repeat region is also believed t o occur during early development, and some experimental evidence indicates that this modification may stabilize the repeats, To study the behavior of full mutation alleles in mitotic cells, we generated human-mouse somatic ce ll hybrids that carry both methylated and unmethylated full mutation FMR1 a lleles, We observed considerable repeat instability and analyzed repeat dyn amics in the hybrids as a function of DNA methylation, repeat length and ce llular differentiation. Our results indicate that although DNA methylation does correlate with stability in primary human fibroblasts, it does not do so in the cell hybrids, Instead, repeat stability in the hybrids is depende nt on repeat length, except in an undifferentiated cellular background wher e large alleles are maintained with a high degree of stability, This stabil ity is lost when the cells undergo differentiation, These results indicate that the determinants of CGG repeat stability are more complex than general ly believed, and suggest an unexpected role for cellular differentiation in this process.