Molecular Nature of Fetal DNA in Maternal Plasma

母体血浆中胎儿 DNA 的分子性质

• 批准号: 7330493
• 负责人: DOROTHY E. LEWIS
• 金额: $ 31.36万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7330493
• 关键词: Acids; Acridine Orange; Annexins; Apoptosis; Apoptotic; Binding; Biological; Biological Assay; Birth; Blood; Blood Circulation; Caring; Cell Nucleus; Cells; Characteristics; Chorionic villi; DNA; DNA Methylation; Data; Detection; Diagnosis; Diagnostic; Diagnostic tests; Down Syndrome; Elements; Fetus; Flow Cytometry; Genes; Genetic; Genetic Materials; Goals; High-Risk Pregnancy; Histones; Image; Invasive; Medical; Methods; Methylation; Modeling; Molecular; Molecular Biology; Nature; Nucleosomes; Physiological; Plasma; Polymerase Chain Reaction; Population Heterogeneity; Pre-Eclampsia; Pregnancy; Pregnant Women; Property; Proteins; Recovery; Risk; Sorting - Cell Movement; Staining method; Stains; System; Technology; Time; Tissues; Variant; Vesicle; Visual; Woman; base; cell type; fetal; human SOX1 protein; human SOX11 protein; human SOX12 protein; human SOX13 protein; human SOX14 protein; human SOX15 protein; human SOX17 protein; human SOX18 protein; human SOX21 protein; human SOX4 protein; human SOX5 protein; human SOX6 protein; human SOX7 protein; human SOX8 protein; improved; in vitro Model; in vivo; interest; keratin CK7; mouse Sox5 protein; mouse Sox7 protein; new technology; novel; prenatal; size; structural biology; trophoblast; tumor

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.

胎儿游离 DNA 存在于母体循环中，通过我们的方法可以实现强劲恢复。血浆中的无细胞胎儿 DNA 甚至比母体血液中完整细胞的 DNA 还要丰富。胎儿 DNA 存在的生物（结构）形式及其在母体血浆中变异的机制尚不清楚。因为人们对这种胎儿遗传物质的诊断效用非常感兴趣，所以有必要进行结构和分子表征，以促进胎儿 DNA 的改进分离和/或富集。 根据我们的初步数据，即母体血浆中存在凋亡小体和 DNA，并且胎儿特定序列可以从含有这些囊泡的流式分选吖啶橙染色血浆级分中富集 3-5 倍，我们假设胎儿​​ DNA 主要以凋亡小体的形式循环。假定的细胞类型是胎盘绒毛（即滋养层细胞）。我们进一步假设胎儿和母体 DNA 的分子特性不同。在这个修订后的提案中，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、形态学和 DNA 染色将用于识别母体血浆中的凋亡小体和核小体，并使用流式细胞术或使用形态学特征对异质细胞群和亚细胞成分进行多参数流成像的新技术 (ImageStreamTM) 进行后续富集。胎儿凋亡小体的鉴定将基于检测细胞凋亡和滋养层的细胞特异性标记物，然后通过实时PCR检测胎儿特异性序列（即SRY基因位点）2）开发诱导细胞凋亡的模型培养系统，用于研究与凋亡小体和凋亡DNA的形成、稳定性和恢复相关的基本特性。 3) 鉴定母体血浆中胎儿DNA的数量和分子形式。将使用多种方法的组合来根据胎儿 DNA 的大小、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。更好地了解母体循环中胎儿 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高风险妊娠评估的方法。

项目成果

Placental release of distinct DNA-associated micro-particles into maternal circulation: reflective of gestation time and preeclampsia.

• DOI: 10.1016/j.placenta.2009.06.012
• 发表时间: 2009-10
• 期刊: PLACENTA
• 影响因子: 3.8
• 作者: Orozco, A. F.;Jorgez, C. J.;Ramos-Perez, W. D.;Popek, E. J.;Yu, X.;Kozinetz, C. A.;Bischoff, F. Z.;Lewis, D. E.
• 通讯作者: Lewis, D. E.

Elevated levels of total (maternal and fetal) beta-globin DNA in maternal blood from first trimester pregnancies with trisomy 21.

21 三体妊娠早期妊娠的母体血液中总（母体和胎儿）β-珠蛋白 DNA 水平升高。

• DOI: 10.1093/humrep/dem154
• 发表时间: 2007
• 期刊: Human reproduction (Oxford, England)
• 作者: Jorgez,CarolinaJ;Dang,DianneD;Wapner,Ronald;Farina,Antonio;Simpson,JoeLeigh;Bischoff,FaridehZ
• 通讯作者: Bischoff,FaridehZ