Characteristics of Fetal Extrachromosomal Circular DNA in Maternal Plasma: Methylation Status and Clearance

Abstract

Background Although the characterization of cell-free extrachromosomal circular DNA (eccDNA) has gained much research interest, the methylation status of these molecules is yet to be elucidated. We set out to compare the methylation densities of plasma eccDNA of maternal and fetal origins, and between small and large molecules. The clearance of fetal eccDNA from maternal circulation was also investigated. Methods We developed a sequencing protocol for eccDNA methylation analysis using tagmentation and enzymatic conversion approaches. A restriction enzyme-based approach was applied to verify the tagmentation results. The efficiency of cell-free fetal eccDNA clearance was investigated by fetal eccDNA fraction evaluations at various postpartum time points. Results The methylation densities of fetal eccDNA (median: 56.3%; range: 40.5–67.6%) were lower than the maternal eccDNA (median: 66.7%; range: 56.5–75.7%) (P = 0.02, paired t-test). In addition, eccDNA molecules from the smaller peak cluster (180–230 bp) were of lower methylation levels than those from the larger peak cluster (300–450 bp). Both of these findings were confirmed using the restriction enzyme approach. We also observed comparable methylation densities between linear and eccDNA of both maternal and fetal origins. The average half-lives of fetal linear and eccDNA in the maternal blood were 30.2 and 29.7 min, respectively. Conclusions We found that fetal eccDNA in plasma was relatively hypomethylated compared to the maternal eccDNA. The methylation densities of eccDNA were positively correlated with their sizes. In addition, fetal eccDNA was found to be rapidly cleared from the maternal blood after delivery, similar to fetal linear DNA.

Small extrachromosomal circular DNA (eccDNA): major functions in evolution and cancer

“… NA [ 114 ] (II) The methylation status of eccDNA in the circulation is different and unique under specific physiological conditions, which makes eccDNA a trace biomarker. Not applicable [ 22 ] NA Not available …”

“…However, the genetic content of eccDNAs shows marked differences among leukocytes from the same individual [16]. Fetal-derived eccDNAs are shorter than maternal eccD-NAs [21], and fetal eccDNAs in plasma are relatively hypomethylated compared with maternal eccDNAs [22]. EccDNAs from tumors have smaller sizes and prefer end coordinates from those derived from normal cells [23][24][25].…”

“…Therefore, the characteristics of tumor-specific eccDNAs in tumor and matched specimens may be helpful for the diagnosis and prognosis of these diseases. Recent studies have shown that the size distributions [107], end locations [108], end motifs [109], and epigenetic features [22] of eccDNAs are helpful for tracing their origin. EccDNAs, including those larger than 2 kb [110], can be released as extracellular free DNAs from healthy and unhealthy cells into biological fluid under different circumstances and may serve as novel biomarkers to shed new insights for the early detection of cancer, the monitoring of responses to drug treatment, and cancer survival [110][111][112][113][114].…”

Extrachromosomal circular DNA: a new potential role in cancer progression

“…The methylation density of eccDNA was positively correlated with its size. In addition, fetal eccDNA was found to be rapidly cleared from maternal blood after delivery, similar to fetal linear DNA[ 113 ]. Unfortunately, the epigenetic nature of eccDNA in cancer has not yet been studied.…”

Cell‐free fetal DNA coming in all sizes and shapes

“…68 They also disappeared from maternal plasma promptly after delivery with apparent half-lives of <1 h, similar to linear cffDNA. 68 Speaking of circular DNA molecules, the mention of mitochon- 69 In such surrogate pregnancies, polymorphic variants could be used to distinguish the fetal mitochondrial molecules from those of the gestational carrier. Notably, more mitochondrial DNA from the gestational carrier circulated in circular forms while the fetal ones were mainly in linear forms.…”