Differentially expressed microRNAs in maternal plasma for the noninvasive prenatal diagnosis of Down syndrome (trisomy 21)

Abstract

Objectives: Most developmental processes are under the control of small regulatory RNAs called microRNAs (miRNAs). We hypothesize that different fetal developmental processes might be reflected by extracellular miRNAs in maternal plasma and may be utilized as biomarkers for the noninvasive prenatal diagnosis of chromosomal aneuploidies. In this proof-of-concept study, we report on the identification of extracellular miRNAs in maternal plasma of Down syndrome (DS) pregnancies.

Methods: Using high-throughput quantitative PCR (HT-qPCR), 1043 miRNAs were investigated in maternal plasma via comparison of seven DS pregnancies with age and fetal sex matched controls.

Results: Six hundred and ninety-five miRNAs were identified. Thirty-six significantly differentially expressed mature miRNAs were identified as potential biomarkers. Hierarchical cluster analysis of these miRNAs resulted in the clear discrimination of DS from euploid pregnancies. Gene targets of the differentially expressed miRNAs were enriched in signaling pathways such as mucin type-O-glycans, ECM-receptor interactions, TGF-beta, and endocytosis, which have been previously associated with DS.

Conclusions: miRNAs are promising and stable biomarkers for a broad range of diseases and may allow a reliable, cost-efficient diagnostic tool for the noninvasive prenatal diagnosis of DS.

Figure 1

Column plots of selected miRNAs representing an expression profile between Down syndrome versus euploid pregnancies.

Figure 2

Clustering based on the global normalized Cqs of the 20 most differentially expressed microRNAs (a) and even clustering based on the raw NRQs of only the 10 most differentially expressed miRNAs (b) were performed. As demonstrated here, clustering can be archived that distinguishes Down syndrome from euploid pregnancies utilizing a subset of 10–20 miRNAs. Sample identification comprised of: sample ID number.fetal sex.week of gestation.group. Abbreviations: f = female, m = male, T = Trisomy 21, N = normal.

Figure 3

Hypothesized mechanisms on the entrance of fetal/placental miRNAs in the maternal circulation. (a) Connection of the mother and fetus via the placenta. (b) Chorionic villi are responsible for sustaining the placenta with nutrients and oxygen. The intervillus space is filled with maternal blood. (c) Cellular release mechanisms and extracellular transportation systems of miRNAs in accordance with Creemers and colleagues [40]. In the cytoplasm, miRNAs can be incorporated into small vesicles, exosomes, which stem from the endosome, and are released from cells when multivesicular bodies coalesce with the plasma membrane. Microvesicles may also be the source of cytoplasmic miRNAs, which are released from the cell via plasma membrane blebbing. miRNAs are also found in circulation in a microparticle-free form. These miRNAs can be associated with high-density lipoproteins or bound to RNA-binding proteins such as Ago2. It remains unknown as to how these miRNA-protein complexes are released from the cell. They have been suggested to be passively released, as by-products of dead cells, or in an active miRNA-specific manner, via interaction with specific membrane channels or proteins.