Chan K C Allen, Ding Chunming, Gerovassili Ageliki, Yeung Sze W, Chiu Rossa W K, Leung Tse N, Lau Tze K, Chim Stephen S C, Chung Grace T Y, Nicolaides Kypros H, Lo Y M Dennis
Department of Chemical Pathology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR.
Clin Chem. 2006 Dec;52(12):2211-8. doi: 10.1373/clinchem.2006.074997. Epub 2006 Oct 26.
We recently demonstrated that the promoter of the RASSF1A gene is hypermethylated in the placenta and hypomethylated in maternal blood cells. This methylation pattern allows the use of methylation-sensitive restriction enzyme digestion for detecting the placental-derived hypermethylated RASSF1A sequences in maternal plasma.
We performed real-time PCR after methylation-sensitive restriction enzyme digestion to detect placental-derived RASSF1A sequences in the plasma of 28 1st-trimester and 43 3rd-trimester pregnant women. We used maternal plasma to perform prenatal fetal rhesus D (RhD) blood group typing for 54 early-gestation RhD-negative women, with hypermethylated RASSF1A as the positive control for fetal DNA detection.
Hypermethylated RASSF1A sequences were detectable in the plasma of all 71 pregnant women. The genotype of plasma RASSF1A after enzyme digestion was identical to the fetal genotype in each case, thus confirming its fetal origin. Nineteen of the 54 pregnant women undergoing prenatal fetal RhD genotyping showed undetectable RHD sequences in their plasma DNA samples. The fetal DNA control, RASSF1A, was not detectable in 4 of the 19 women. Subsequent chorionic villus sample analysis revealed that 2 of these 4 women with negative RHD and RASSF1A signals were in fact carrying RhD-positive fetuses.
Hypermethylated RASSF1A is a universal marker for fetal DNA and is readily detectable in maternal plasma. When applied to prenatal RhD genotyping, this marker allows the detection of false-negative results caused by low fetal DNA concentrations in maternal plasma. This new marker can also be applied to many other prenatal diagnostic and monitoring scenarios.
我们最近证明，RASSF1A基因的启动子在胎盘中高度甲基化，而在母体血细胞中低甲基化。这种甲基化模式使得可以利用甲基化敏感的限制性内切酶消化来检测母体血浆中胎盘来源的高度甲基化的RASSF1A序列。
我们在甲基化敏感的限制性内切酶消化后进行实时PCR，以检测28例孕早期和43例孕晚期孕妇血浆中胎盘来源的RASSF1A序列。我们使用母体血浆对54例妊娠早期RhD阴性妇女进行产前胎儿恒河猴D（RhD）血型分型，将高度甲基化的RASSF1A作为胎儿DNA检测的阳性对照。
在所有71例孕妇的血浆中均可检测到高度甲基化的RASSF1A序列。酶消化后血浆RASSF1A的基因型在每种情况下均与胎儿基因型相同，从而证实了其胎儿来源。在54例接受产前胎儿RhD基因分型的孕妇中，有19例在其血浆DNA样本中未检测到RHD序列。在这19名妇女中，有4名未检测到胎儿DNA对照RASSF1A。随后的绒毛取样分析显示，这4名RHD和RASSF1A信号均为阴性的妇女中有2名实际上怀有RhD阳性胎儿。
高度甲基化的RASSF1A是胎儿DNA的通用标志物，并且很容易在母体血浆中检测到。当应用于产前RhD基因分型时，该标志物可以检测到由于母体血浆中胎儿DNA浓度低而导致的假阴性结果。这种新标志物也可应用于许多其他产前诊断和监测情况。
