Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.