Discovery and validation of GNA12^circle as a first-trimester plasma eccDNA marker for early-onset preeclampsia

Early-onset preeclampsia (EOPE) is a major cause of maternal and perinatal morbidity and is characterized by placental dysfunction, systemic endothelial injury, and hypertensive vascular stress. Because hypertensive disorders of pregnancy may also signal later maternal cardiovascular and cerebrovascular vulnerability, effective biomarkers for first-trimester risk assessment remain clinically important. Extrachromosomal circular DNA (eccDNA), a stable form of circulating cell-free DNA, has emerged as a potential source of disease-associated biomarkers. This study aimed to characterize first-trimester plasma eccDNA alterations associated with subsequent EOPE and to identify and validate a candidate circulating eccDNA marker for early risk assessment.

A two-stage nested case-control study was conducted within a prospective birth cohort. In the discovery stage, plasma samples collected at 11-13 weeks of gestation from 5 women who subsequently developed EOPE and 5 matched normotensive controls were profiled by Circle-Seq to characterize genome-wide eccDNA alterations. Candidate eccDNAs were prioritized through differential abundance analysis and were further confirmed by outward PCR and Sanger sequencing. In the validation stage, the candidate selected marker was quantified by junction-specific qPCR in an independent cohort of 109 EOPE cases and 109 controls. Its potential predictive value was further evaluated alone and in combination with routine first-trimester clinical variables.

In the exploratory discovery analysis, 410 nominally differentially abundant candidate eccDNAs were identified as a hypothesis-generating pool. Among these, GNA12^circle (chr7:2876332-2,876,692) was prioritized and experimentally validated at the circular junction. In the independent validation cohort, plasma GNA12^circle levels were significantly higher in women who later developed EOPE than in controls. When combined with routine first-trimester variables, GNA12^circle improved predictive performance. The RF model showed the best overall cross-validated performance among the evaluated classifiers, with a mean held-out test-fold AUC of 0.843.

First-trimester plasma eccDNA profiling revealed distinct alterations associated with subsequent EOPE, from which GNA12^circle was identified and validated as a candidate circulating marker. These findings support further investigation of circulating eccDNA for early EOPE risk assessment in larger multicenter populations.