The Quantitative Analysis Of Fetal RASSF1A Sequences In Maternal Plasma Between Normal Pregnancy And Pre-eclampsia

Objective:To investigate the feasibility of quantitative detection of hypermethylated Ras association domain family 1A (RASSFIA) gene in maternal plasma by TaqMan probe real-time PCR and analysis the RASSFIA levels before and after methylation-sensitive restriction enzymes digestion. The aims of the detection of hypermethylated RASSFIA gene in maternal plasma from all three trimesters of pregnancy were to show its feature of cell free fetal DNA and to make up defieients of genetie markers. This study also aimed at investigating of its application value in pre-eclampsia.Methods:80 pregnant women(7-41 gestational weeks) including pregnancy of first trimester (20 cases), second trimester (20 cases), third trimester (20 cases), mild pre-eclampsia (10 cases) and severe pre-eclampsia (10 cases) were selected as study groups,20 normal non-pregnant women were selected as control group. Free DNA of plasma samples was extracted, The methylation-sensitive restriction enzymes were specifically cutting the maternally derived background hypomethylated sequences but leaving the intact hypermethylated sequences, RASSFIA levels before and after double methylation-sensitive restriction enzymes digestion of HinP1I and Hhal were determined using fluorescence quantitative polymerase chain reaction (FQ-PCR) to measure total and fetal cell-free DNA, respectively. At the same time,β-actin gene was detected as a control to confirm complete enzymes digestion.Results:(1) Hypermethylated RASSFIA sequences in maternal plasma were detected in 77 of 80 pregnant women, and were not detected in the plasma of non-pregnant women. They can be detected in maternal plasma as early as at 7+3 th week of gestation. The sensitivity and specificity of fetal gene detection by post-digestion RASSFIA measurement were 96.25% and 100%. (2) The concentrations of hypermethylated RASSFIA in maternal plasma increased as pregnancy progresses, the mean concentrations of hypermethylated RASSFIA gene were 55.52copies/ml in first trimester,112.46copies/ml in second trimester,557.50copies/ml in third trimester, respectively. We futher observed the post-digestion RASSFIA concentrations as a fraction of the total RASSFIA concentrations obtained without enzymes digestion RASSFIA which corresponded to1.78%,2.91%, and 9.45% in the three trimesters. (3) The mean concentrations of hypermethylated RASSFIA gene were 3.72-fold higher in maternal plasma of pre-eclamptic pregnancies than in controls. There was positive correlation between fetal-derived hypermethylated RASSFIA levels and the severity of pre-eclampsia. (4) The post-digestion RASSFIA was completely cleared at 24h after delivery in 10 pregnant women of the third trimester, it still can be detectable in 8 of 10 pre-eclamptic pregnant women, which was cleared after a week.Conclusion:Hypermethylated RASSFIA gene may be considered as a epigenetic marker to detect the fetal DNA in maternal plasma. It is easy and precise to detect the hypermethylated RASSFIA gene in maternal plasma by TaqMan probe real-time PCR. Our study shows the correlation of maternal plasma hypermethylaed RASSFIA concentrations with pre-eclampsia. The elevated amounts of maternal plasma hypermethylaed RASSFIA can forecast the process and prognosis of pre-eclampsia and eclampsia. The quantitative detection of the placental epigenetic signature of the RASSFIA gene in maternal plasma expand the clinic application of cell free fetal DNA in noninvasive prenatal diagnosis.