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Single-cell Epigenetic Errors Can Be Uncovered and Rescued, Study Shows

NEW YORK (GenomeWeb News) – Using a new single-cell DNA methylation analysis technique, researchers were able to detect DNA methylation errors in mice, they reported in Science today. Errors in methylation and other epigenetic changes have been linked to a number of human syndromes, the researchers added.

In this study, once the researchers probed a number of early stage mouse embryos at different loci and found such methylation errors, they endeavored to correct them through pronuclear transfer.

"The single-cell DNA-methylation assay is a powerful tool to address such defects and is well suited for accurate diagnosis in these patients or to address the occurrence of rare, random imprinting defects suspected to result from assisted reproductive technology," the research team, led by Chanchao Lorthongpanich and Lih Feng Cheow from Singapore's Agency for Science, Technology and Research, or A*Star, wrote.

Genomic imprinting is thought to be established through a two-step process, part of which occurs in the germline and part of which takes place in the pre-implantation embryo. Researchers have hypothesized that epimutations occur when mechanisms to preserve differentially methylated regions — mechanisms that work through proteins like DNMT1, PGC7/STELLA, ZFP57, and TRIM28 — break down. Changes to the epigenetic state early in development could to lead to chimeras and, possibility, developmental arrest.

To analyze the DNA methylation of single cells, the researchers combined multiplex real-time qPCR with a methylation-sensitive restriction digest in a microfluidic device. Using this, they focused on six imprinted loci. They validated their device on control oocytes, finding that paternally imprinted regions were unmethylated while maternally imprinted ones were methylated.

Lorthongpanich, Cheow, and their colleagues noted that early stages of embryonic development rely on maternal genes. Turning to maternal mice with and without the scaffolding protein gene Trim28, which helps maintain imprinting, the researchers found that nearly all of the control blastomeres had methylated alleles while the Trim28-null embryos had varying levels of hypomethylation at all of the loci being tested.

"[T]he loss of DNA methylation occurred randomly and independently at similar average rates across all cells," the investigators said.

Still, they calculated that the degree of demethylation was lower than predicted, indicating that there was incomplete penetrance. Other factors like PGC7/STELLA and, to a lesser degree, DNMT1, they suggested, could be protecting the methylation status of the loci.

"Despite incomplete penetrance, examining only six of 21 known germline [differentially methylated regions] reveals the prodigious potential for imprinting defect combinations, the phenotypic outcome of which will further depend on blastomere viability, and their contribution to the embryo proper," the researchers said. "This mosaicism may account for phenotypic traits, such as occasional hemi-anophthalmia in maternal-null Trim28 fetuses, which are hard to explain by simple genetics."

TRIM28, they added, appeared to be necessary immediately following fertilization.

To try to fix such early epigenetic defects, the researchers turned to pronuclear transfer. They moved maternal Trim218-null pronuclei into enucleated control zygotes. Within an hour, they reported, the zygotes displayed nuclear TRIM28, and 17 percent of the embryos developed into mouse pups.

"These 'rescue pups' became fertile adults and showed normal H19 DMR methylation (the most frequently affected imprinted locus in maternal-null Trim28 mutants) in tail biopsies, comparable to controls and in contrast to maternal-null embryos," the researchers said.

Such a pronuclear transfer approach, Lorthongpanich, Cheow, and their colleagues added, could be developed to prevent epimutation-based imprinting syndromes.

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