NIPD Genetics, Nicosia, Cyprus.
Mol Genet Genomic Med. 2020 Feb;8(2):e1094. doi: 10.1002/mgg3.1094. Epub 2019 Dec 10.
Non-invasive prenatal testing (NIPT) for fetal aneuploidies has rapidly been incorporated into clinical practice. Current NGS-based methods can reliably detect fetal aneuploidies non-invasively with fetal fraction of at least 4%. Inaccurate fetal fraction assessment can compromise the accuracy of the test as affected samples with low fetal fraction have an increased risk for misdiagnosis. Using a novel set of fetal-specific differentially methylated regions (DMRs) and methylation sensitive restriction digestion (MSRD), we developed a multiplex ddPCR assay for accurate detection of fetal fraction in maternal plasma.
We initially performed MSRD followed by methylation DNA immunoprecipitation (MeDIP) and NGS on fetal and non-pregnant female tissues to identify fetal-specific DMRs. DMRs with the highest methylation difference between the two tissues were selected for fetal fraction estimation employing MSRD and multiplex ddPCR. Chromosome Y multiplex ddPCR assay (YMM) was used as a reference standard, to develop our fetal fraction estimation model in male pregnancy samples. Additional 123 samples were tested to examine whether the model is sex dependent and/or ploidy dependent.
In all, 93 DMRs were identified of which seven were selected for fetal fraction estimation. Statistical analysis resulted in the final model which included four DMRs (FFMM). High correlation with YMM-based fetal fractions was observed using 85 male pregnancies (r = 0.86 95% CI: 0.80-0.91). The model was confirmed using an independent set of 53 male pregnancies.
By employing a set of well-characterized DMRs, we developed a SNP-, sex- and ploidy-independent methylation-based multiplex ddPCR assay for accurate fetal fraction estimation.
胎儿非整倍体的无创产前检测（NIPT）已迅速纳入临床实践。目前基于 NGS 的方法可以可靠地无创检测胎儿非整倍体，其胎儿分数至少为 4%。不准确的胎儿分数评估会影响测试的准确性，因为受影响的低胎儿分数样本有更高的误诊风险。我们使用一组新的胎儿特异性差异甲基化区域（DMR）和甲基化敏感限制性消化（MSRD），开发了一种用于准确检测母体血浆中胎儿分数的多重 ddPCR 检测方法。
我们最初在胎儿和非妊娠女性组织上进行了 MSRD 随后进行了甲基化 DNA 免疫沉淀（MeDIP）和 NGS，以鉴定胎儿特异性 DMR。选择两种组织之间甲基化差异最大的 DMR 用于胎儿分数估计，采用 MSRD 和多重 ddPCR。我们使用染色体 Y 多重 ddPCR 检测（YMM）作为参考标准，在男性妊娠样本中建立我们的胎儿分数估计模型。另外测试了 123 个样本，以检验该模型是否依赖于性别和/或倍性。
总共鉴定了 93 个 DMR，其中 7 个被用于胎儿分数估计。统计分析得出了最终模型，其中包括四个 DMR（FFMM）。使用 85 例男性妊娠的 YMM 基础胎儿分数进行了高相关性分析（r=0.86，95%置信区间：0.80-0.91）。使用 53 例独立的男性妊娠对该模型进行了验证。
通过使用一组特征明确的 DMR，我们开发了一种 SNP-、性别和倍性独立的基于甲基化的多重 ddPCR 检测方法，用于准确估计胎儿分数。
