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: We next examined whether the DNA sequence was altered at the replication initiation site. Therefore, we sequenced a 1-kb DNA segment containing the SNP and MRE1b element in FXS hESCs and control hESCs (Fig. 2 A). The analysis revealed that in FXS hESCs, SI-214 and WCMC37 and in FXS fetal fibroblast GM07072, the SNP variant C was found at the replication initiation site instead of the SNP variant T. We observed no other noteworthy alteration in the DNA sequences close to the SNP site 53 kb upstream of the repeats and the FMR1 gene. In addition, we determined the number of AGG interruptions in the CGG repeats in nonaffected and FXS cells. Control cells H14, H9, and GM00011 contained two AGG interruptions, whereas FXS hESC SI-214 contained one, and FXS hESC WCMC37 and FXS fetal fibroblast GM07072 had none. These results are in agreement with previous analysis of AGG interruptions in cells from patients in whom loss of AGG interruptions increases the risk of CGG repeat expansion (Yrigollen et al., 2012; Nolin et al., 2013). Collectively, we found that the FXS cells studied here contain the SNP variant C.
Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461 firstname.lastname@example.org email@example.com.