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Objectives    RASSF1A  has been described to be differentially methylated between fetal and maternal DNA and can therefore be used as a universal sex-independent marker to confirm the presence of fetal sequences in maternal plasma. However, this requires highly sensitive methods. We have previously shown that Pyrophosphorolysis-activated Polymerization (PAP) is a highly sensitive technique that can be used in noninvasive prenatal diagnosis. In this study, we have used PAP in combination with bisulfite conversion to develop a new universal methylation-based assay for the detection of fetal methylated  RASSF1A  sequences in maternal plasma.    Methods  Bisulfite sequencing was performed on maternal genomic (g)DNA and fetal gDNA from chorionic villi to determine differentially methylated regions in the  RASSF1A  gene using bisulfite specific PCR primers. Methylation specific primers for PAP were designed for the detection of fetal methylated  RASSF1A  sequences after bisulfite conversion and validated.    Results  Serial dilutions of fetal gDNA in a background of maternal gDNA show a relative percentage of ∼3% can be detected using this assay. Furthermore, fetal methylated  RASSF1A  sequences were detected both retrospectively as well as prospectively in all maternal plasma samples tested (n = 71). No methylated  RASSF1A  specific bands were observed in corresponding maternal gDNA. Specificity was further determined by testing anonymized plasma from non-pregnant females (n = 24) and males (n = 21). Also, no methylated  RASSF1A  sequences were detected here, showing this assay is very specific for methylated fetal DNA. Combining all samples and controls, we obtain an overall sensitivity and specificity of 100% (95% CI 98.4%–100%).    Conclusions  Our data demonstrate that using a combination of bisulfite conversion and PAP fetal methylated  RASSF1A  sequences can be detected with extreme sensitivity in a universal and sex-independent manner. Therefore, this assay could be of great value as an addition to current techniques used in noninvasive prenatal diagnostics.

Mrassf1a-Pap, a Novel Methylation-Based Assay for the Detection of Cell-Free Fetal DNA in Maternal Plasma