Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation.
Bottom Line: This origin is absent in FXS human embryonic stem cells (hESCs), which have the SNP variant C, but present in the nonaffected hESCs, which have a T variant.Interestingly, premutation hESCs have a replication origin and the T variant similar to nonaffected hESCs.These results suggest that a T/C SNP located at a replication origin could contribute to the inactivation of this replication origin in FXS hESCs, leading to altered replication fork progression through the repeats, which could result in repeat expansion to the FXS full mutation.
Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461 firstname.lastname@example.org email@example.com.Show MeSH
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Mentions: Next, we asked whether the premutation hESCs, which contain a T, have a similar replication program to nonaffected hESCs. Nonaffected hESCs contain the SNP variant T and an active replication origin 53 kb upstream of the repeats (Gerhardt et al., 2014). This is in contrast to FXS hESCs, which contain a C and the replication origin is missing. First, we tested whether the CGG repeats in the premutation hESCs containing the SNP variant T expand further in cell culture. Therefore, we analyzed the repeat length in multiple passages of the premutation hESC WCMC5 by Southern blotting over the course of several months (Fig. 4 A). We observed no large changes in the repeat length in these cell passages. We also analyzed cell passages by Asuragen PCR analysis. All passages contained the same repeat length (73 repeats). We concluded that the repeats in the premutation hESCs WCMC5 do not expand further in cell culture.
Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461 firstname.lastname@example.org email@example.com.