No abstract

Preimplantation genetic diagnosis (PGD) is widely applied in reciprocal translocation carriers to increase the chance for a successful live birth. However, reciprocal translocation carrier embryos were seldom discriminated from the normal ones mainly due to the technique restriction. Here we established a clinical applicable approach to identify precise breakpoint of reciprocal translocation and to further distinguish normal embryos in PGD. In the preclinical phase, rearrangement breakpoints and adjacent single nucleotide polymorphisms (SNPs) were characterized by next-generation sequencing following microdissecting junction region (MicroSeq) from 8 reciprocal translocation carriers. Junction-spanning PCR and sequencing further discovered precise breakpoints. The precise breakpoints were identified in 7/8 patients and we revealed that translocations in 6 patients caused 9 gene disruptions. In the clinical phase of embryo analysis, informative SNPs were chosen for linkage analyses combined with PCR analysis of the breakpoints to identify the carrier embryos. From 15 blastocysts diagnosed to be chromosomal balanced, 13 blastocysts were identified to be carriers and 2 to be normal. Late prenatal diagnoses for five carriers and one normal fetus confirmed the carrier diagnosis results. Our results suggest that MicroSeq can accurately evaluate the genetic risk of translocation carriers and carrier screen is possible in later PGD treatment.

High telomerase activity is a characteristic of human embryonic stem cells (hESCs), however, the regulation and maintenance of correct telomere length in hESCs is unclear. In this study we investigated telomere elongation in hESCs in vitro and found that telomeres lengthened from their derivation in blastocysts through early expansion, but stabilized at later passages. We report that the core unit of telomerase, hTERT, was highly expressed in hESCs in blastocysts and throughout long-term culture; furthermore, this was regulated in a Wnt-b-catenin-signaling-dependent manner. Our observations that the alternative lengthening of telomeres (ALT) pathway was suppressed in hESCs and that hTERT knockdown partially inhibited telomere elongation, demonstrated that high telomerase activity was required for telomere elongation. We observed that chromatin modification through trimethylation of H3K9 and H4K20 at telomeric regions decreased during early culture. This was concurrent with telomere elongation, suggesting that epigenetic regulation of telomeric chromatin may influence telomerase function. By measuring telomere length in 96 hESC lines, we were able to establish that telomere length remained relatively stable at 12.0261.01 kb during later passages (15-95). In contrast, telomere length varied in hESCs with genomic instability and hESC-derived teratomas. In summary, we propose that correct, stable telomere length may serve as a potential biomarker for genetically stable hESCs.