Tong Yu K, Ding Chunming, Chiu Rossa W K, Gerovassili Ageliki, Chim Stephen S C, Leung Tak Y, Leung Tse N, Lau Tze K, Nicolaides Kypros H, Lo Y M Dennis
Department of Chemical Pathology, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China.
Clin Chem. 2006 Dec;52(12):2194-202. doi: 10.1373/clinchem.2006.076851. Epub 2006 Oct 13.
The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. However, the use of maternal plasma fetal DNA for the direct detection of fetal chromosomal aneuploidies has not been reported. We postulate that the aneuploidy status of a fetus could be revealed by an epigenetic allelic ratio approach, i.e., by analyzing the allelic ratio of a single-base variation present within DNA molecules exhibiting a placental-specific epigenetic signature in maternal plasma.
Placental-derived fetal-specific unmethylated maspin (SERPINB5) promoter sequences on human chromosome 18 were detectable in placental-maternal DNA mixtures and in maternal plasma by bisulfite modification followed by methylation-specific PCR (MSP) and primer extension. The ratios between the extension products of the 2 alleles were calculated for heterozygous placentas, placental-maternal blood cell DNA mixtures, and maternal plasma samples. The allelic ratios were compared between pregnancies carrying trisomy 18 and euploid fetuses.
The epigenetic allelic ratios of all tested trisomy 18 samples deviated from the reference range obtained from euploid samples (placental DNA, 1.135 to 2.052; placental-maternal DNA mixtures, 1.170 to 1.985; maternal plasma, 0.330 to 3.044; without skew correction on the raw mass spectrometric data). A theoretical model was established and validated that predicted that a minimum of 200 copies of genomic DNA after bisulfite conversion were required for distinguishing euploid and aneuploid fetuses with confidence.
Epigenetic allelic ratio analysis of maternal plasma DNA represents a promising approach for noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.
母体血浆中游离胎儿DNA的发现为无创产前诊断开辟了新的可能性。然而，尚未见利用母体血浆胎儿DNA直接检测胎儿染色体非整倍性的报道。我们推测，胎儿的非整倍性状态可通过表观遗传等位基因比率法揭示，即通过分析母体血浆中呈现胎盘特异性表观遗传特征的DNA分子内单碱基变异的等位基因比率。
通过亚硫酸氢盐修饰，随后进行甲基化特异性PCR（MSP）和引物延伸，在胎盘-母体DNA混合物和母体血浆中可检测到人18号染色体上胎盘来源的胎儿特异性未甲基化maspin（SERPINB5）启动子序列。计算杂合胎盘、胎盘-母体血细胞DNA混合物和母体血浆样本中两个等位基因延伸产物之间的比率。比较怀有18三体胎儿和整倍体胎儿的妊娠之间的等位基因比率。
所有检测的18三体样本的表观遗传等位基因比率均偏离从整倍体样本获得的参考范围（胎盘DNA，1.135至2.052；胎盘-母体DNA混合物，1.170至1.985；母体血浆，0.330至3.044；原始质谱数据未经偏斜校正）。建立并验证了一个理论模型，该模型预测亚硫酸氢盐转化后至少需要200个基因组DNA拷贝才能可靠地区分整倍体和非整倍体胎儿。
母体血浆DNA的表观遗传等位基因比率分析是一种很有前景的胎儿染色体非整倍性无创产前诊断方法。
