Department of Clinical Genetics and GROW School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, the Netherlands;
Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands.
Clin Chem. 2015 Dec;61(12):1515-23. doi: 10.1373/clinchem.2015.244962. Epub 2015 Oct 14.
Noninvasive genetic tests that use cell-free fetal DNA (cffDNA) are used increasingly in prenatal care. A low amount of cffDNA can have detrimental effects on the reliability of these tests. A marker to confirm the presence of fetal nucleic acids is therefore required that is universally applicable and easy to incorporate.
We developed a novel multiplex, single-tube, noninvasive fetal sex determination assay by combining amplification of AMELY cffDNA with one-step reverse transcription (RT)-PCR of trophoblast-derived cell-free RNA (cfRNA), which functions as a sex-independent fetoplacental marker. We tested plasma samples from 75 pregnant women in duplicate in a blinded fashion. The fetus was considered to be male in the case of a positive result for AMELY and cfRNA amplification in both RT-PCRs. The fetus was considered to be female in the case of negative AMELY and positive cfRNA result in both RT-PCRs. In other cases, the test was repeated. We compared the results with invasive prenatal testing and pregnancy outcomes.
The AMELY cffDNA amplification and cfRNA result was unambiguous and identical in duplicate in 71 of 75 plasma samples (95%). Four samples (5%) required an extra replicate because of an absent fetoplacental marker. Thereafter, fetal sex was correctly determined in all 75 plasma samples.
Amplification of trophoblast-derived cfRNA is a reliable marker for the confirmation of the presence of fetoplacentally derived nucleic acids in noninvasive fetal sex determination.
使用游离胎儿 DNA（cffDNA）的非侵入性基因检测在产前护理中越来越多地被使用。少量的 cffDNA 会对这些测试的可靠性产生不利影响。因此，需要一种普遍适用且易于纳入的确认胎儿核酸存在的标志物。
我们通过结合 AMELY cffDNA 的扩增和滋养层来源的无细胞 RNA（cfRNA）的一步逆转录（RT）-PCR，开发了一种新颖的多重、单管、非侵入性胎儿性别鉴定测定法，cfRNA 作为一种与性别无关的胎胎盘标志物。我们以盲法对 75 名孕妇的血浆样本进行了重复测试。如果 AMELY 和 cfRNA 扩增的 RT-PCR 均为阳性，则认为胎儿为男性。如果 AMELY 为阴性且 cfRNA 结果为阳性，则认为胎儿为女性。在其他情况下，重复进行测试。我们将结果与侵入性产前检测和妊娠结局进行了比较。
75 份血浆样本中的 71 份（95%）在重复检测中 AMELY cffDNA 扩增和 cfRNA 结果明确且一致。由于缺乏胎胎盘标志物，有 4 份样本（5%）需要额外重复检测。此后，所有 75 份血浆样本中的胎儿性别均得到了正确鉴定。
滋养层来源的 cfRNA 扩增是确认非侵入性胎儿性别鉴定中胎胎盘来源核酸存在的可靠标志物。
