Abstract
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.MethodsBisulfite 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.ResultsSerial 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%).ConclusionsOur 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.
Highlights
Over the past years, the use of cell-free fetal DNA for noninvasive prenatal diagnosis (NIPD) has proven its clinical potential in a wide range of fields
Our data demonstrate that using a combination of bisulfite conversion and Pyrophosphorolysis-activated Polymerization (PAP) fetal methylated RASSF1A sequences can be detected with extreme sensitivity in a universal and sex-independent manner
This assay could be of great value as an addition to current techniques used in noninvasive prenatal diagnostics
Summary
The use of cell-free fetal DNA (cffDNA) for noninvasive prenatal diagnosis (NIPD) has proven its clinical potential in a wide range of fields. The possibilities for using cffDNA in NIPD are numerous, they do require highly sensitive and specific techniques to detect the low levels of fetal sequences in the pool of maternal plasma DNA early in gestation. Additional detection of paternally inherited sequences could be used to discriminate between a true negative result in case of a female pregnancy, or a false negative result in case of low levels of circulating cffDNA. These methods are quite laborious since both biological parents need to be tested along with the plasma sample and not all SNPs and loci tested will be informative. A large panel of different markers need to be tested for each individual case [5]
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