Fully Expanded FMR1CGG Repeats Exhibit a Length-and Differentiation-Dependent Instability in Cell Hybrids That is Independent of DNA Methylation

Abstract

The fragile X syndrome is characterized at the molecular level by expansion and methylation of a CGG trinucleotide repeat located within the FMR1 locus. The tissues of most full mutation carriers are mosaic for repeat size, but these mutational patterns tend to be well conserved when comparing multiple tissues within an individual. Moreover, full mutation alleles are stable in cultured fibroblasts. These observations have been used to suggest that fragile X CGG repeat instability normally is limited to a period during early embryogenesis. DNA methylation of the repeat region is also believed to 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 cell hybrids that carry both methylated and unmethylated full mutation FMR1 alleles. We observed considerable repeat instability and analyzed repeat dynamics in the hybrids as a function of DNA methylation, repeat length and cellular 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 dependent on repeat length, except in an undifferentiated cellular background where large alleles are maintained with a high degree of stability. This stability is lost when the cells undergo differentiation. These results indicate that the determinants of CGG repeat stability are more complex than generally believed, and suggest an unexpected role for cellular differentiation in this process.

Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells

“…Length-dependent modifiers of repeat instability are common (32)(33)(34)(35). To explore whether ZFN-induced instability depends on the length of the repeat tract, we transfected the zfGCT nuclease into APRT(CAG) 61 CHO cells and human HPRT(CAG) 68 cells.…”

Paroxysmal Nocturnal Hemoglobinuria: Stem Cells and Clonality

“…DNA repair is progressively attenuated during the process of cellular differentiation, 25 possibly leading to differentiation-dependent spontaneous mutations. 26 PIG-A mutations occurring in cells that lack self-renewal capacity (e.g., CFC) will not propagate disease since the average lifespan of these cells is roughly 125 days. 27 This explains why PIG-A mutations in more differentiated cells (e.g., granulocytes) are common in healthy control subjects; however, these data do not explain how clonal expansion of a PIG-A mutant stem cell occurs in patients with PNH.…”

CpG Methylation Modifies the Genetic Stability of Cloned Repeat Sequences

“…Completely and partially methylated CGG tracts as short as 20 to 100 repeats have also been observed (Allingham-Hawkins et al 1996;Tassone et al 1999;Genc et al 2000). It has been suggested that for both germline and somatic tissues, the absence of CpG methylation may enhance the deletion of (CGG)n repeats (Burman et al 1999;Helderman-van den Enden et al 1999;Salat et al 2000;Wohrle et al 2001). However, it is not clear how methylation might contribute to CGG instability.…”

“…Alterations in CpG methylation is a candidate modifier of primate repeat stability because many unstable elements are part of or are embedded within large CpG islands. Furthermore, the instability of certain repeats is restricted to specific loci (Richards et al 1996), specific tissues (Anvret et al 1993;Malter et al 1997), or differentiation status (Burman et al 1999;Wohrle et al 2001), or instability occurs only during specific developmental stages (Malter et al 1997;Martorell et al 1997). Because CpG methylation is highly regulated in a tissue-and development-specific manner (Razin and Shemer 1995), its alteration may contribute to repeat instability.…”

“…Evidence supporting this notion comes from apparent differences in genetic stability of the methylated and unmethylated expanded (CGG)n repeat of fragile X (Malter et al 1997;Wohrle et al 1998;Burman et al 1999;Wohrle et al 2001) and the hypermethylation associated with large expansions of the myotonic dystrophy (CTG)n repeat . Hyper-and hypomethylation in certain tumors is associated with microsatellite instabilities (Herman et al 1998;Toyota et al 1999) and large deletions (Makos et al 1993).…”