母体外周血游离 DNA 大规模平行测序检测胎儿三体及单基因病：概念验证研究。
Detection of fetal trisomy and single gene disease by massively parallel sequencing of extracellular vesicle DNA in maternal plasma: a proof-of-concept validation.
机构信息
BGI-Shenzhen, Beishan Industrial Zone, Shenzhen, 518083, China.
China National GeneBank, BGI-Shenzhen, Shenzhen, 518120, China.
出版信息
BMC Med Genomics. 2019 Nov 4;12(1):151. doi: 10.1186/s12920-019-0590-8.
BACKGROUND
During human pregnancy, placental trophectoderm cells release extracellular vesicles (EVs) into maternal circulation. Trophoblasts also give rise to cell-free DNA (cfDNA) in maternal blood, and has been used for noninvasive prenatal screening for chromosomal aneuploidy. We intended to prove the existence of DNA in the EVs (evDNA) of maternal blood, and compared evDNA with plasma cfDNA in terms of genome distribution, fragment length, and the possibility of detecting genetic diseases.
METHODS
Maternal blood from 20 euploid pregnancies, 9 T21 pregnancies, 3 T18 pregnancies, 1 T13 pregnancy, and 2 pregnancies with FGFR3 mutations were obtained. EVs were separated from maternal plasma, and confirmed by transmission electronic microscopy (TEM), western blotting, and flow cytometry (FACS). evDNA was extracted and its fetal origin was confirmed by quantitative PCR (qPCR). Pair-end (PE) whole genome sequencing was performed to characterize evDNA, and the results were compared with that of cfDNA. The fetal risk of aneuploidy and monogenic diseases was analyzed using the evDNA sequencing data.
RESULTS
EVs separated from maternal plasma were confirmed with morphology by TEM, and protein markers of CD9, CD63, CD81 as well as the placental specific protein placental alkaline phosphatase (PLAP) were confirmed by western blotting or flow cytometry. EvDNA could be successfully extracted for qPCR and sequencing from the plasma EVs. Sequencing data showed that evDNA span on all 23 pairs of chromosomes and mitochondria, sharing a similar distribution pattern and higher GC content comparing with cfDNA. EvDNA showed shorter fragments yet lower fetal fraction than cfDNA. EvDNA could be used to correctly determine fetal gender, trisomies, and de novo FGFR3 mutations.
CONCLUSIONS
We proved that fetal DNA could be detected in EVs separated from maternal plasma. EvDNA shared some similar features to plasma cfDNA, and could potentially be used to detect genetic diseases in fetus.
背景
在人类妊娠期间，胎盘滋养层细胞将细胞外囊泡（EVs）释放到母体外周循环中。滋养层细胞也会在母体外周血中释放无细胞 DNA（cfDNA），并已被用于非侵入性产前筛查染色体非整倍体。我们旨在证明母体外周血 EVs（evDNA）中存在 DNA，并比较 evDNA 与血浆 cfDNA 在基因组分布、片段长度和检测遗传疾病的可能性方面的差异。
方法
收集了 20 例正常妊娠、9 例 21 三体妊娠、3 例 18 三体妊娠、1 例 13 三体妊娠和 2 例 FGFR3 基因突变妊娠的母体外周血。通过透射电子显微镜（TEM）、western blot 和流式细胞术（FACS）证实从母体外周血浆中分离出 EVs。提取 evDNA，并通过 qPCR 确认其胎儿来源。进行 PE 全基因组测序以表征 evDNA，并将结果与 cfDNA 进行比较。使用 evDNA 测序数据分析非整倍体和单基因疾病的胎儿风险。
结果
通过 TEM 观察到分离自母体外周血浆的 EVs 的形态，通过 western blot 或 FACS 检测到 CD9、CD63、CD81 等蛋白标志物以及胎盘特异性蛋白碱性磷酸酶（PLAP）。可以从血浆 EVs 中成功提取 evDNA 进行 qPCR 和测序。测序数据显示，evDNA 覆盖了所有 23 对染色体和线粒体，与 cfDNA 相比，分布模式相似，GC 含量更高。evDNA 片段较短，但胎儿分数比 cfDNA 低。evDNA 可用于正确判断胎儿性别、三体和新发 FGFR3 突变。
结论
我们证明了可以从母体外周血浆中分离的 EVs 中检测到胎儿 DNA。evDNA 与血浆 cfDNA 具有一些相似的特征，并且可能可用于检测胎儿的遗传疾病。
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