Massively parallel sequencing of maternal cell-free DNA (cfDNA) is widely used to test fetal genetic abnormalities in non-invasive prenatal testing (NIPT). However, sequencing-based approaches are still of high cost. Building upon previous knowledge that placenta, the main source of fetal circulating DNA, is hypomethylated in comparison to maternal tissue counterparts of cfDNA, we propose that targeting either unmodified or 5-hydroxymethylated CG sites specifically enriches fetal genetic material and reduces numbers of required analytical sequencing reads thereby decreasing cost of a test. We employed uTOPseq and hmTOP-seq approaches which combine covalent derivatization of unmodified or hydroxymethylated CG sites, respectively, with next generation sequencing, or quantitative real-time PCR. We detected increased 5-hydroxymethylcytosine (5hmC) levels in fetal chorionic villi (CV) tissue samples as compared with peripheral blood. Using our previously developed uTOP-seq and hmTOP-seq approaches we obtained whole-genome uCG and 5hmCG maps of 10 CV tissue and 38 cfDNA samples in total. Our results indicated that, in contrast to conventional whole genome sequencing, such epigenomic analysis highly specifically enriches fetal DNA fragments from maternal cfDNA. While both our approaches yielded 100% accuracy in detecting Down syndrome in fetuses, hmTOP-seq maintained such accuracy at ultra-low sequencing depths using only one million reads. We identified 2164 and 1589 placenta-specific differentially modified and 5-hydroxymethylated regions, respectively, in chromosome 21, as well as 3490 and 2002 Down syndrome-specific differentially modified and 5-hydroxymethylated regions, respectively, that can be used as biomarkers for identification of Down syndrome or other epigenetic diseases of a fetus. uTOP-seq and hmTOP-seq approaches provide a cost-efficient and sensitive epigenetic analysis of fetal abnormalities in maternal cfDNA. The results demonstrated that T21 fetuses contain a perturbed epigenome and also indicated that fetal cfDNA might originate from fetal tissues other than placental chorionic villi. Robust covalent derivatization followed by targeted analysis of fetal DNA by sequencing or qPCR presents an attractive strategy that could help achieve superior sensitivity and specificity in prenatal diagnostics.

中文翻译：

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用于非侵入性产前检测的母体游离 DNA 中胎儿未修饰和 5-羟甲基化 CG 位点的鉴定

母体无细胞 DNA (cfDNA) 的大规模平行测序广泛用于在无创产前检测 (NIPT) 中检测胎儿遗传异常。然而，基于测序的方法成本仍然很高。基于先前的知识，胎盘是胎儿循环 DNA 的主要来源，与 cfDNA 的母体组织对应物相比甲基化程度低，我们建议靶向未修饰或 5-羟甲基化的 CG 位点专门丰富胎儿遗传物质并减少所需的分析测序数量读取从而降低测试成本。我们采用了 uTOPseq 和 hmTOP-seq 方法，它们分别将未修饰或羟甲基化 CG 位点的共价衍生化与下一代测序或定量实时 PCR 相结合。与外周血相比，我们检测到胎儿绒毛膜绒毛 (CV) 组织样本中 5-羟甲基胞嘧啶 (5hmC) 水平升高。使用我们之前开发的 uTOP-seq 和 hmTOP-seq 方法，我们总共获得了 10 个 CV 组织和 38 个 cfDNA 样本的全基因组 uCG 和 5hmCG 图。我们的结果表明，与传统的全基因组测序相比，这种表观基因组分析高度特异性地富集了来自母体 cfDNA 的胎儿 DNA 片段。虽然我们的两种方法在检测胎儿唐氏综合症方面都达到了 100% 的准确度，但 hmTOP-seq 在超低测序深度下仅使用 100 万次读取就保持了这种准确度。我们分别在 21 号染色体中鉴定了 2164 个和 1589 个胎盘特异性差异修饰区域和 5-羟甲基化区域，以及分别为 3490 和 2002 唐氏综合征特异性差异修饰和 5-羟甲基化区域，可用作鉴定唐氏综合征或其他胎儿表观遗传疾病的生物标志物。uTOP-seq 和 hmTOP-seq 方法为母体 cfDNA 中的胎儿异常提供了经济高效且灵敏的表观遗传分析。结果表明，T21 胎儿包含一个扰动的表观基因组，还表明胎儿 cfDNA 可能来自胎盘绒毛膜以外的胎儿组织。稳健的共价衍生化，然后通过测序或 qPCR 对胎儿 DNA 进行靶向分析，是一种有吸引力的策略，有助于在产前诊断中实现卓越的灵敏度和特异性。可用作鉴定唐氏综合症或其他胎儿表观遗传疾病的生物标志物。uTOP-seq 和 hmTOP-seq 方法为母体 cfDNA 中的胎儿异常提供了经济高效且灵敏的表观遗传分析。结果表明，T21 胎儿包含一个扰动的表观基因组，还表明胎儿 cfDNA 可能来自胎盘绒毛膜以外的胎儿组织。稳健的共价衍生化，然后通过测序或 qPCR 对胎儿 DNA 进行靶向分析，是一种有吸引力的策略，有助于在产前诊断中实现卓越的灵敏度和特异性。可用作鉴定唐氏综合症或其他胎儿表观遗传疾病的生物标志物。uTOP-seq 和 hmTOP-seq 方法为母体 cfDNA 中的胎儿异常提供了经济高效且灵敏的表观遗传分析。结果表明，T21 胎儿包含一个扰动的表观基因组，还表明胎儿 cfDNA 可能来自胎盘绒毛膜以外的胎儿组织。稳健的共价衍生化，然后通过测序或 qPCR 对胎儿 DNA 进行靶向分析，是一种有吸引力的策略，有助于在产前诊断中实现卓越的灵敏度和特异性。结果表明，T21 胎儿包含一个扰动的表观基因组，还表明胎儿 cfDNA 可能来自胎盘绒毛膜以外的胎儿组织。稳健的共价衍生化，然后通过测序或 qPCR 对胎儿 DNA 进行靶向分析，是一种有吸引力的策略，有助于在产前诊断中实现卓越的灵敏度和特异性。结果表明，T21 胎儿包含一个扰动的表观基因组，还表明胎儿 cfDNA 可能来自胎盘绒毛膜以外的胎儿组织。稳健的共价衍生化，然后通过测序或 qPCR 对胎儿 DNA 进行靶向分析，是一种有吸引力的策略，有助于在产前诊断中实现卓越的灵敏度和特异性。