Molecular Nature of Fetal DNA in Maternal Plasma

无细胞胎儿DNA存在于母体循环中，通过我们的方法可以强劲复苏。血浆中的无细胞胎儿DNA甚至比来自母体血液中完整细胞的DNA更丰富。胎儿DNA存在的生物学(结构)形式及其在母体血浆中变化的机制尚不清楚。由于人们对这种胎儿遗传物质的诊断效用非常感兴趣，因此有必要进行结构和分子表征，以促进改进胎儿DNA的分离和/或浓缩。 根据我们的初步数据，孕妇血浆中存在凋亡小体和DNA，并且从含有这些小泡的流动分选的丫啶橙染色的血浆部分中可以浓缩3-5倍的胎儿特定序列，我们假设胎儿DNA主要以凋亡小体的形式循环。推测细胞类型为胎盘绒毛(即滋养层细胞)。我们进一步假设胎儿和母体的DNA在分子性质上是不同的。在这个修订的提案中，我们的项目目标是：1)确定孕妇血浆中循环胎儿DNA的结构性质。视觉、形态和DNA染色将被用来识别母体血浆中的凋亡小体和核小体，并用于随后使用流式细胞术或利用形态特征开发的用于多参数流动成像的新技术(ImageStreamTM)进行浓缩。胎儿凋亡小体的鉴定将基于细胞凋亡和滋养层细胞特异性标志物的检测，然后通过实时荧光聚合酶链式反应检测胎儿特异性序列(即SRY基因位点)2)建立诱导细胞凋亡的模型培养系统，以研究与凋亡小体和凋亡DNA的形成、稳定和恢复相关的基本性质。3)鉴定孕妇血浆中胎儿DNA存在的数量和分子形式(S)。将结合多种方法，根据大小、单链或双链动机的存在、组蛋白的稳定性和DNA甲基化来表征和量化胎儿DNA。更好地了解孕妇循环中胎儿DNA的生物学和分子特征将有助于改进非侵入性产前基因诊断和评估高危妊娠的方法。

Cell-free fetal DNA is present in the maternal circulation and by our methods amenable to robust recovery. Cell-free fetal DNA in plasma is even more abundant than DNA derived from intact cells in maternal blood. The biological (structural) form in which fetal DNA exists and the mechanisms underlying its variation in maternal plasma are unclear. Because great interest lies in the diagnostic utility of this fetal genetic material, structural and molecular characterization to facilitate improved isolation and/or enrichment of fetal DNA is warranted. Based on our preliminary data that apoptotic bodies and DNA are present in maternal plasma and that fetal specific sequences can be enriched 3-5 fold from flow sorted acridine orange stained plasma fractions containing these vesicles, we hypothesize that fetal DNA circulates predominantly in the form of apoptotic bodies. The presumptive cell type is placental villi (i.e. trophoblasts). We further hypothesize that fetal and maternal DNA differ in their molecular properties. In this revised proposal, our project aims are: 1) to determine the structural nature of circulating fetal DNA in plasma from pregnant women. Visual, morphologic and DNA-staining will be used to identify apoptotic bodies and nucleosomes in maternal plasma, and for subsequent enrichment using either flow cytometry or a novel technology developed for multiparametric flow imaging of a heterogeneous population of cells and sub-cellular elements using morphologic features (ImageStreamTM). Identification of fetal apoptotic bodies will be based on detection of cellular specific markers for apoptosis and trophoblasts followed by real-time PCR to detect fetal specific sequences (i.e. SRY gene locus) 2) To develop a model culture system to induce apoptosis, for studying fundamental properties associated with formation, stability and recovery of apoptotic bodies and apoptotic DNA. 3) To identify the quantity and molecular form(s) in which fetal DNA exists in maternal plasma. A combination of approaches will be used to characterize and quantify fetal DNA on the basis of size, existence of single or double stranded motives, stabilization by histones, and DNA methylation. Better understanding of the biology and molecular characteristics of fetal DNA in maternal circulation will enable improved methods for non-invasive prenatal genetic diagnosis and assessment of high risk pregnancies.