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Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation.

Gerhardt J, Zaninovic N, Zhan Q, Madireddy A, Nolin SL, Ersalesi N, Yan Z, Rosenwaks Z, Schildkraut CL - J. Cell Biol. (2014)

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461 jeannine.gerhardt@gmail.com carl.schildkraut@einstein.yu.edu.

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FXS hESCs contain the SNP variant C instead of T and a methylation pattern surrounding the SNP that is similar to the DNA methylation pattern in nonaffected hESCs. (A) Table shows that DNA sequence analysis of 1-kb genomic DNA containing the SNP revealed that nonaffected hESCs H14 and H9 and fetal fibroblast GM00011 contain the SNP variant T and FXS hESCs SI-214 and WCMC37 and FXS fetal fibroblast GM07072 contain SNP variant C. The repeat sizes and AGG interruptions were determined by Asuragen PCR analysis. (B) DNA methylation pattern of the 367-bp genomic DNA segment containing the SNP was analyzed in nonaffected hESCs H14 and H9 and fetal fibroblast GM00011 and FXS hESCs SI-214 and WCMC37 and FXS fetal fibroblast GM07072. For each cell line, 10 DNA methylation analyses were performed at the genomic DNA segment including the SNP (containing 10 CpG sites and two CpA sites). A white circle indicates <10% CpG methylation, and gray circles indicate if <50% CpG methylation was detected. Filled black circles mark the positions of ≥50% methylated CpG sites.
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fig2: FXS hESCs contain the SNP variant C instead of T and a methylation pattern surrounding the SNP that is similar to the DNA methylation pattern in nonaffected hESCs. (A) Table shows that DNA sequence analysis of 1-kb genomic DNA containing the SNP revealed that nonaffected hESCs H14 and H9 and fetal fibroblast GM00011 contain the SNP variant T and FXS hESCs SI-214 and WCMC37 and FXS fetal fibroblast GM07072 contain SNP variant C. The repeat sizes and AGG interruptions were determined by Asuragen PCR analysis. (B) DNA methylation pattern of the 367-bp genomic DNA segment containing the SNP was analyzed in nonaffected hESCs H14 and H9 and fetal fibroblast GM00011 and FXS hESCs SI-214 and WCMC37 and FXS fetal fibroblast GM07072. For each cell line, 10 DNA methylation analyses were performed at the genomic DNA segment including the SNP (containing 10 CpG sites and two CpA sites). A white circle indicates <10% CpG methylation, and gray circles indicate if <50% CpG methylation was detected. Filled black circles mark the positions of ≥50% methylated CpG sites.

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.


Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation.

Gerhardt J, Zaninovic N, Zhan Q, Madireddy A, Nolin SL, Ersalesi N, Yan Z, Rosenwaks Z, Schildkraut CL - J. Cell Biol. (2014)

FXS hESCs contain the SNP variant C instead of T and a methylation pattern surrounding the SNP that is similar to the DNA methylation pattern in nonaffected hESCs. (A) Table shows that DNA sequence analysis of 1-kb genomic DNA containing the SNP revealed that nonaffected hESCs H14 and H9 and fetal fibroblast GM00011 contain the SNP variant T and FXS hESCs SI-214 and WCMC37 and FXS fetal fibroblast GM07072 contain SNP variant C. The repeat sizes and AGG interruptions were determined by Asuragen PCR analysis. (B) DNA methylation pattern of the 367-bp genomic DNA segment containing the SNP was analyzed in nonaffected hESCs H14 and H9 and fetal fibroblast GM00011 and FXS hESCs SI-214 and WCMC37 and FXS fetal fibroblast GM07072. For each cell line, 10 DNA methylation analyses were performed at the genomic DNA segment including the SNP (containing 10 CpG sites and two CpA sites). A white circle indicates <10% CpG methylation, and gray circles indicate if <50% CpG methylation was detected. Filled black circles mark the positions of ≥50% methylated CpG sites.
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Related In: Results  -  Collection

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fig2: FXS hESCs contain the SNP variant C instead of T and a methylation pattern surrounding the SNP that is similar to the DNA methylation pattern in nonaffected hESCs. (A) Table shows that DNA sequence analysis of 1-kb genomic DNA containing the SNP revealed that nonaffected hESCs H14 and H9 and fetal fibroblast GM00011 contain the SNP variant T and FXS hESCs SI-214 and WCMC37 and FXS fetal fibroblast GM07072 contain SNP variant C. The repeat sizes and AGG interruptions were determined by Asuragen PCR analysis. (B) DNA methylation pattern of the 367-bp genomic DNA segment containing the SNP was analyzed in nonaffected hESCs H14 and H9 and fetal fibroblast GM00011 and FXS hESCs SI-214 and WCMC37 and FXS fetal fibroblast GM07072. For each cell line, 10 DNA methylation analyses were performed at the genomic DNA segment including the SNP (containing 10 CpG sites and two CpA sites). A white circle indicates <10% CpG methylation, and gray circles indicate if <50% CpG methylation was detected. Filled black circles mark the positions of ≥50% methylated CpG sites.
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.

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461 jeannine.gerhardt@gmail.com carl.schildkraut@einstein.yu.edu.

Show MeSH
Related in: MedlinePlus