基于亲子细胞系和凋亡机制的无创产前检测游离DNA参考物质的研究

Non-invasive prenatal testing via cell-free DNA (cfDNA) in the plasma of pregnant mothers is particularly valuable in clinical practices. Reference materials (RMs) are required to ensure accurate results. However, since it is not feasible to obtain enough clinical samples from the plasma of pregnant women with specific genetic diseases, simulated samples are now being considered to be alternatives. Ideal cfDNA reference materials are required to represent the features of nucleosomal DNA and positioning of maternal and fetal cfDNA generated by apoptosis and maternal–fetal genetic relations. No RMs with the two characters are reported. In the previous study, we have creatively prepared cfDNA RMs for somatic mutation detection in cancer by micrococcal nuclease (MNase) digestion. This study aims to establish good-quality RMs that biologically and genetically resemble authentic cfDNA in pregnant mothers for the detection of fetal aneuploidy and single gene disorders with cfDNA, based on parent-child cell lines and apoptosis-associated enzymatic digestion with MNase, Exonulease III (ExoIII) and DNA fragmentation factor 40 (DFF 40), by means of CRISPR/Cas9 technology, digital PCR and next-generation sequencing (NGS). Our study will establish a new platform for preparation of cfDNA reference materials for noninvasive genetic tests, which are believed to greatly promote the standardizations of non-invasive prenatal testing in clinical practices.

孕妇外周血游离DNA(cfDNA)无创产前检测展示了广阔的临床应用价值，要保证检测的准确性，必须有可靠的参考物质，但源于孕妇血浆具有特定遗传异常的临床样本不可能大量获得，因此主要通过模拟样本来建立。理想的参考物质，母体和胎儿cfDNA应具有凋亡产生的核小体单体为主的片段分布特征和遗传关系，目前未见满足上述特征的参考物质报道。在前期研究中，我们创造性地采用微球菌核酸酶（MNase）建立了肿瘤cfDNA参考物质。本研究拟在现有基础上，使用亲子细胞系和基于凋亡机制的MNase/核酸外切酶III (ExoIII)/ DNA片段化因子40 (DFF40)消化，采用CRISPR/Cas9、数字PCR和高通量测序等技术，建立最接近真实样本生物和遗传特征的染色体非整倍体和单基因遗传病无创产前检测cfDNA参考物质。本研究将为无创产前检测参考物质的研制提供新的技术平台，并推动无创产前检测的标准化。

孕妇外周血游离DNA（cfDNA）无创产前检测展示了广阔的临床应用价值，要保证检测的准确性，必须有可靠的参考物质，但源于孕妇血浆具有特定遗传异常的临床样本不可能大量获得，因此主要通过模拟样本来建立。理想的参考物质，母体和胎儿cfDNA应具有凋亡产生的核小体单体为主的片段分布特征和遗传关系。采用DNA片段化因子（DNA fragmentation factor, DFF）和MNase分别模拟母体和胎儿游离DNA片段，建立了基于亲子细胞系的最大程度接近真实临床样本的无创产前参考物质。通过对模拟样本与真实样本进行深度测序，比较二核苷酸组成分布、核小体保护序列长度鉴定及转录起始位点（transcription start site, TSS）区域核小体占有率等，证实模拟样本与真实样本具有一致的核小体定位模式及cfDNA片段化特征，并具有方法广泛适用性。研究成果“一种应用于无创产前检测的参考物质及其制备方法”已申请国际PCT专利，进入美国国家专利受理阶段；发表在临床检验排名第一的Clinical Chemistry杂志，同期刊登由阿姆斯特丹自由大学医学中心Erik A. Sistermans教授发表Editorials，认为“该研究是未来无创产前检测和诊断非常重要的一个进步”。本团队通过原材料制备、流程体系建立、性能评价标准等三个方面的研究，现已形成NIPT参考物质产品，应用于我国无创产前检测室间质量评价，并完成技术成果转化。本研究解决了无创产前检测试剂方法建立、性能确认、临床实验室检测质量控制参考物质来源问题，对促进无创产前实验室标准化具有重要意义。

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