Feto-Maternal DNA/RNA Trafficking:Biology & Applications

描述(由申请者提供)：拟议项目的总体目标是了解无细胞DNA和RNA贩运的生物学，并利用所获得的知识开发新的产前诊断应用。有待检验的总体假设是，孕妇循环和羊水中的无细胞胎儿DNA和mRNA来自特定的胎儿组织来源，并包含独特的胎儿基因序列，可用于遗传和发育障碍的产前诊断。到目前为止，证据表明，大量的无细胞DNA，以及较少程度的RNA，在孕妇的血清和血浆中循环。这些核酸的半衰期很短，在胎儿或新生儿出生后几个小时内就会被清除。羊水中也含有大量的无细胞胎儿DNA和RNA。目前，人们对这些核酸序列的起源组织以及它们是如何产生的知之甚少。该项目的具体目标1是利用母体血浆、存档血清样本和羊水上清液中的无细胞胎儿DNA作为临床材料来源，开发新的临床检测方法，以改进现有的非侵入性分析，以确定胎儿非整倍体或单基因疾病的风险，如先天性肾上腺增生(CAH)。到目前为止，临床应用的重点是检测和量化独特的胎儿序列，以此作为妊娠并发症的指示。一个主要的限制是，这些分析中的许多仅限于携带Y染色体的男性胎儿。因此，特定的目标2是使用独特的胎儿mRNA基因序列来鉴定和定量孕妇血浆中的胎儿核酸，而不受胎儿性别的影响。我们将检验通过母体血液中循环的mRNA序列测量的基因表达增加或减少是否可以用于非侵入性检测胎儿非整倍体或其他妊娠并发症。在目标3中，我们将通过扩增造血、胎盘和胎儿来源的特定基因来确定循环核酸的来源组织。我们还将利用羊水中存在的无细胞mRNA通过微阵列来检测基因表达。我们将检查由于胎龄和不同病理因素造成的胎儿之间的差异。

DESCRIPTION (provided by applicant): The overall goal of the proposed project is to understand the biology of feto-maternal cell-free DNA and RNA trafficking, and to use the knowledge gained to develop novel prenatal diagnostic applications. The overall hypothesis to be tested is that cell-free fetal DNA and mRNA in the maternal circulation and amniotic fluid originates from specific fetal tissue sources and contains unique fetal gene sequences that can be used for prenatal diagnosis of genetic and developmental disorders. Evidence thus far indicates that significant amounts of cell-free DNA, and to a lesser degree, RNA, circulate within the serum and plasma of pregnant women. These nucleic acids have a short half-life and are cleared within hours of delivery of the fetus or newborn. There is also a significant amount of cell-free fetal DNA and RNA present in the amniotic fluid. Currently, little is known about the tissue of origin of these nucleic acid sequences and how they are produced. Specific aim 1 of the project is to use cell-free fetal DNA in maternal plasma, archived serum samples, and amniotic fluid supernatants as sources of clinical material to develop new clinical tests that could improve existing noninvasive assays to determine the risk of fetal aneuploidy or single gene disorders such as congenital adrenal hyperplasia (CAH). To date clinical applications have focused on detection and quantitation of uniquely fetal sequences as an indication of pregnancy complications. A major limitation has been that many of these analyses have been limited to male fetuses, which carry a Y chromosome. Thus, specific aim 2 is to use uniquely fetal mRNA gene sequences for the identification and quantitation of fetal nucleic acids in maternal plasma, independent of fetal gender. We will examine if increased or decreased gene expression, as measured by circulating mRNA sequences in maternal blood, can be used to noninvasively detect fetal aneuploidy or other complications of pregnancy. In aim 3 we will determine the tissue of origin of the circulating nucleic acids by amplifying specific genes of hematopoietic, placental, and fetal origin. We will also use cell-free mRNA present in amniotic fluid to examine gene expression via microarrays. We will examine differences between fetuses due to gestational age and different pathologies.