Early non-invasive epigenetic approach for assessing trisomy risk in maternal plasma

Karyotyping is the standard confirmatory test for identifying chromosomal abnormalities, such as Trisomy 21, which requires amniotic fluid from pregnant women. The present study investigated the potential of methylated cell-free DNA (mcf-DNA) and methylated extracellular vesicle-derived DNA (mev-DNA) as an early, non-invasive epigenetic approach, specifically focusing on fetal-specific methylated regions (FSMRs) of RASSF1A, ERG, and UMODL1 (U1 and U2) to assess Trisomy 21 risk in maternal plasma.

Blood samples were collected from pregnant women (n = 120) between 10th and 24th weeks of gestation who were at higher risk for Trisomy 21. Of the 120 women, 3 were found to be positive for Trisomy 21 through karyotyping method. Moreover, mcf-DNA and mev-DNA were isolated from Trisomy-positive and age-matched healthy pregnant women (n = 8) and non-pregnant women (n = 8). FSMRs were analyzed using qPCR to compare the cycle threshold (Ct) values. Gene copy number analysis was performed using the plasmid standards method to assess Trisomy 21 detection sensitivity.

Trisomy pregnancies had significantly lower mean Ct values for RASSF1A and UMODL1 (U1 and U2) in both mcf-DNA and mev-DNA than healthy pregnancies, which was further confirmed by higher copy numbers in trisomy pregnancies than in healthy pregnancies. Moreover, the gene copy number in mcf-DNA was significantly higher than that in mev-DNA for the RASSF1A, ERG, and UMODL1 (U1 and U2) genes in trisomy pregnancies.

This pilot study demonstrates the feasibility of using mcf-DNA and mev-DNA for detecting Trisomy 21-associated fetal methylation signatures in maternal plasma. While consistent with earlier findings, these results validate the applicability of methylated DNA immunoprecipitation (MeDIP)-based qPCR methylation assays in a North Indian cohort and support their potential integration into population-specific non-invasive prenatal screening strategies.