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Circular Cell-Free DNA in Plasma of Pregnant Women May Serve as Novel Biomarker

NEW YORK – Circular cell-free DNA of both maternal and fetal origin can be identified in the plasma of pregnant women, according to a new study, pointing to a possible new type of biomarker for noninvasive prenatal testing.

Linear cell-free DNA from maternal plasma has long formed the basis of NIPT for fetal aneuploidies. While extrachromosomal circular DNA (eccDNA) in plasma — which could be more resistant to exonucleases and therefore more stable than linear circulating DNA — has been observed in mice as well as in people before, it has not been reported in pregnant women.

Dennis Lo of the Chinese University of Hong Kong and his colleagues isolated and sequenced eccDNA molecules from the plasma of pregnant women to find that those of fetal origin are generally shorter than those of maternal origin, a result they reported in the Proceedings of the National Academy of Sciences this week.

"EccDNA in maternal plasma with their increased biostability and distinct molecular signatures might add to the toolbox of the rapidly developing field of noninvasive prenatal testing," they wrote in their paper.

For their study, the researchers analyzed plasma DNA from five pregnant women. After enriching for circular DNA, they linearized and sequenced the eccDNA molecules. 

Using maternal- and fetal-specific SNPs, the researchers then assigned the eccDNA as being of maternal or fetal origin. Fetal-origin eccDNA molecules were generally shorter than those of maternal origin, averaging  around 202 base pairs in size, while maternal eccDNA averaged around 338 base pairs. This, the researchers noted, was consistent with what has been observed in linear fetal and maternal cell-free DNA. 

However, the MspI digestion for linearization they relied on might have affected the portion of eccDNAs that could be analyzed. To confirm their findings, they also used a tagmentation-based approach to isolate and analyze eccDNAs from another set of five pregnant women.

This identified significantly more eccDNA molecules than the restriction enzyme-based approach, and again, fetal eccDNA was smaller than maternal eccDNA.

By mapping the eccDNAs back to the genome, the researchers found they were enriched in 5'-untranslated, exonic, and CpG island regions, indicating that circular DNAs are not randomly generated from the genome. 

There were also recurrent patterns within eccDNAs: both the start and end positions are flanked by pairs of trinucleotide segments with four-base spacers. Further analysis of these patterns hinted that homologous recombination and microhomology-mediated end joining could serve as the mechanisms through which eccDNAs are formed from double-stranded DNA.

However, the researchers noted that both their restriction enzyme and tagmentation-based approaches work selectively on double-stranded DNA, which may have led them to underestimate the presence of single-stranded eccDNAs. 

EccDNA molecules from maternal plasma could ultimately serve as biomarkers, they suggested, with the added potential benefit of circular DNA being more robust than linear DNA. 

"It would be interesting for future studies to explore the potential aberrations of maternal plasma eccDNA profiles in different pregnancy-associated disorders, such as preeclampsia and preterm birth," Lo and his colleagues wrote.