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: Because we did not detect a major difference in the DNA methylation pattern at the replication initiation site, we asked whether the T instead of a C at the SNP site in FXS cells was associated with repeat instability in FXS hESCs. To test this, we derived a few additional hESC lines from embryos of premutation carriers: WCMC4 (male normal), WCMC5 (male premutation), and WCMC13 (female premutation; Fig. 3 A and Fig. S2; Colak et al., 2014). We first examined the repeat size by Southern blotting (Fig. 3 B) and further confirmed our findings by the AmplideX PCR assay (Asuragen; Fig. S3). WCMC4 has a normal repeat size (30 repeats with two AGG interruptions; Fig. S3 A and Fig. 3 B), whereas WCMC5 and 13 contain the premutation repeat size with 73 and 70 CGG repeats, respectively, and have no AGG interruptions (Fig. S3, B and C; and Fig. 3 B). We determined the DNA sequence at and surrounding the SNP. Similar to the nonaffected cells, WCMC4, 5, and 13 contain the SNP variant T (Fig. 3 C) in contrast to the FXS hESC line WCMC37, which contains the C variant. Because of the privacy rights of the donors of human embryos, the haplotype structure of the premutation female parent cannot be established. In summary, we obtained two premutation hESCs, neither of which had AGG interruptions nor the SNP variant C in the MRE1b element.
Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461 firstname.lastname@example.org email@example.com.