Development of a novel MSRE-dPCR assay for non-invasive prenatal testing of Trisomy 21

Abstract

Currently, next-generation sequencing (NGS)-based non-invasive prenatal diagnosis testing (NIPT) of fetal T21 has been proven to be much more advantageous than traditional serum biochemical tests in terms of accuracy and sensitivity. Nevertheless, serum biochemical tests remain the first choice for the screening of trisomy 21 (T21) since NGS is too expensive and time-consuming. As an alternative, this paper proposes methylation-sensitive restriction endonuclease (MSRE)-quantitative polymerase chain reaction (qPCR)/digital PCR (dPCR) methods for the screening of T21, which enrich the fraction of fetal-specific DNA by digesting maternal sequences and could more easily reflect the fold changes of chromosomes 21. For this purpose, 64 DMRs on chromosome 21 and the control chromosome were tested for their ability to detect T21 with MSRE-qPCR/dPCR. After MSRE digestion, 8 chromosome 21-specific DMRs and a chromosome 1-reference DMR (CD48) exhibited significant differences between fetal and maternal DNA, which were then applied for multi-index detection via qPCR or dPCR. After testing 24 simulated samples, the corresponding calculation formulas were established for MSRE-qPCR (5-marker panel) and MSRE-dPCR (4-marker panel), and the distinguishing accuracies were 87.5% and 100%, indicating the better performance of MSRE-dPCR. Finally, the detection limit of MSRE-dPCR was found to be 2.44-4.76%, and the established method could identify T21 or healthy pregnancies using cffDNA with an accuracy of 18/19. In general, a novel strategy was developed to screen possible DMRs and to establish MSRE-dPCR methods for NIPT of T21 pregnancies. Future work would focus on identifying more suitable DMRs and establishing better prediction methods with more clinical samples.