Identifying Differentially Methylated Sites for Methylation-Sensitive qPCR-based NIPT of Trisomies 13 and 18

Trisomies 13 and 18 are severe chromosomal disorders with high neonatal mortality rates. Noninvasive prenatal testing based on next-generation sequencing provides accurate detection of these aneuploidies but remains costly. This study investigates methylation-sensitive quantitative PCR (MS-qPCR) as a cost-effective alternative to Next-generation sequencing (NGS)-based NIPT.

Differentially methylated sites (DMSs) were selected from prior studies based on their trisomy/disomy ratios and validated using methylation-sensitive restriction enzymes. Optimized primers for chromosomes 13 and 18 were tested on chorionic villus sampling (CVS) and peripheral blood (PBL) DNA. qPCR parameters were optimized to achieve high efficiency and reproducibility.

Twenty-two DMSs out of 50 targets with significant trisomy/disomy ratios were successfully validated. Optimal amplification efficiencies (1.85-2.10) were achieved, with primers exhibiting high specificity and reproducibility. Also, the methylation-sensitive restriction enzymes with the highest trisomy/disomy ratios for each selected DMS are reported.

MS-qPCR represents a promising alternative to NGS for NIPT, offering a scalable, cost-effective solution for detecting trisomies 13 and 18. Future research should validate these findings using cell-free DNA (cfDNA) in maternal plasma and expand testing to diverse populations.