BACKGROUND Due to technical issues related to cell-specific capture methods, amplification, and sequencing, noninvasive prenatal testing (NIPT) based on fetal nucleated red blood cells (fNRBCs) has rarely been used for the detection of monogenic disorders. METHODS Maternal peripheral blood was collected from 11 families with hereditary hearing loss. After density gradient centrifugation and cellular immunostaining for multiple biomarkers, candidate individual fetal cells were harvested by micromanipulation and amplified by whole-genome amplification (WGA). Whole-exome sequencing/whole-genome sequencing (WGS) and Sanger sequencing were performed on the identified fNRBCs to determine the fetal genotype. The impact of single-cell and pooled WGA products on the sequencing quality and results was compared. A combined analysis strategy, encompassing whole-exome sequencing/WGS, haplotype analysis, and Sanger sequencing, was used to enhance the NIPT results. RESULTS fNRBCs were harvested and identified in 81.8% (9/11) of families. The results of cell-based-NIPT (cb-NIPT) were consistent with those of invasive prenatal diagnosis in 8 families; the coincidence rate was 88.9% (8/9). The combined analysis strategy improved the success of cb-NIPT. The overall performance of pooled WGA products was better than that of individual cells. Due to a lack of alternative fetal cells or sufficient sequencing data, cb-NIPT failed in 3 families. CONCLUSIONS We developed a novel fNRBC-based NIPT method for monogenic disorders. By combining multiple analysis strategies and multiple fetal cell WGA products, the problem of insufficient genome information in a single cell was remedied. Our method has promising prospects in the field of NIPT for the detection of monogenic disorders.

中文翻译：

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基于胎儿有核红细胞的单基因疾病新型无创产前检测方法的探索。

背景技术由于与细胞特异性捕获方法、扩增和测序相关的技术问题，基于胎儿有核红细胞(fNRBC)的无创产前检测(NIPT)很少用于检测单基因疾病。方法采集11个遗传性听力损失家系的母亲外周血。经过密度梯度离心和多种生物标志物的细胞免疫染色后，通过显微操作收获候选个体胎儿细胞并通过全基因组扩增（WGA）进行扩增。对鉴定出的 fNRBC 进行全外显子组测序/全基因组测序（WGS）和桑格测序以确定胎儿基因型。比较了单细胞和混合 WGA 产品对测序质量和结果的影响。采用包括全外显子组测序/WGS、单倍型分析和桑格测序的组合分析策略来增强 NIPT 结果。结果 在 81.8% (9/11) 的家庭中收获并鉴定了 fNRBC。8个家庭基于细胞的NIPT（cb-NIPT）结果与侵入性产前诊断结果一致；符合率为 88.9% (8/9)。组合分析策略提高了 cb-NIPT 的成功率。混合 WGA 产品的整体性能优于单个细胞。由于缺乏替代胎儿细胞或足够的测序数据，3 个家庭的 cb-NIPT 失败。结论 我们针对单基因疾病开发了一种基于 fNRBC 的新型 NIPT 方法。通过结合多种分析策略和多种胎儿细胞WGA产品，解决了单细胞基因组信息不足的问题。我们的方法在单基因疾病检测的 NIPT 领域具有广阔的前景。