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Quake Team Publishes Results of Circulating RNA-Seq Experiments in Pregnant Women

NEW YORK (GenomeWeb) – Using sequencing to profile circulating RNA in maternal blood shows promise as a tool for monitoring pregnancy and fetal development, according to a study published yesterday in the Proceedings of the National Academy of Sciences, by Stephen Quake and colleagues at Stanford University.

"We demonstrate that it is possible to track specific longitudinal phenotypic changes in both the mother and the fetus and that it is possible to directly measure transcripts from a variety of fetal tissues in the maternal blood sample," the authors wrote.

In addition, the researchers showed that the non-invasive gene expression profiling method might also hold potential as a diagnostic for Alzheimer's disease and other neurologic diseases.

Quake and his team previously developed a method for using sequencing to analyze circulating fetal DNA in maternal blood samples, which has since been commercialized as a non-invasive prenatal aneuploidy test by Verinata Health, now owned by Illumina.

In the PNAS study this week, Quake and his co-authors reported on sequencing not DNA, but RNA in maternal blood, demonstrating that their method could distinguish between fetal and maternal contributions to circulating RNA, as well as measure changes in the levels of different gene transcripts over the course of pregnancy.

The group initially looked at four non-pregnant subjects to assess the ability of sequencing to specifically target RNA. The researchers extracted RNA from the subjects' plasma, converting it to cDNA, amplifying it, and sequencing the products.

In this initial experiment, the team was able to show that most of the resulting reads originated from RNA transcripts and not potentially contaminating DNA. The researchers also showed that they could match cell-free RNA molecules in these non-pregnant subjects to their origin in different tissue types.

Then, analyzing RNA from a cohort of 11 pregnant women, the team demonstrated that they could distinguish RNA contributed by the maternal genome from that expression of the fetal genome by analyzing about 100 genes that encode transcripts containing paternal SNPs distinct from the fetus' maternal inheritance.

The team quantified the relative fetal contribution to circulating RNA in these 11 women, which they reported increased, in general, as the pregnancies progressed and then decreased after delivery.

They also identified non-coding transcripts, specifically circular RNAs, which appeared to be expressed specifically during different trimesters. According to the authors, detecting these non-coding RNAs "may improve our ability to monitor the health of the mother and fetus."

Overall, the group also saw the numbers of genes expressed increase across ascending trimesters, followed by a steep drop off after delivery.

One of the most distinct temporal trends, the team reported, was exhibited by the chorionic gonadotropin family of RNA transcripts. The protein beta human chorionic gonadotropin has been previously shown to peak during the first trimester, the authors wrote. In their RNA-seq data, the researchers saw that the cell-free transcriptome mirrored this pattern.

"The fact that the RNA transcript levels show the same trends as the protein supports the notion of using RNA as a practical biomarker in situations where it is not straightforward to detect the protein," the authors wrote.

Two other transcripts, MARCH2 and RAB6B, showed similar temporal trends, they reported.

Interested in looking at RNA expressed specifically from other fetal tissue types, the researchers developed a set of quantitative PCR assays focusing on placental, fetal brain, and fetal liver-associated transcripts.

Using the same qPCR approach, the group also decided to look at RNA from the blood of six Alzheimer's patients and 10 age-matched healthy controls. According to the authors, patients with the disease showed significantly higher levels of the APP transcript, which encodes a precursor molecule implicated in the development of amyloid plaques.

Based on this hint, the authors wrote that cell-free RNA might be considered as a potential blood-based diagnostic for Alzheimer's disease and other neurodegenerative disorders.

"We anticipate these results are a stepping stone toward translating the temporal dynamics of plasma mRNA for clinical diagnosis of pregnancy-associated complications and developmental diseases, especially those that are temporal in nature and involve cellular apoptosis," they wrote

Joined by many others in the field of non-invasive fetal DNA sequencing, Quake and his colleagues are also not alone in advancing to RNA-seq of the fetal fraction of maternal blood.

A team led by Chinese University of Hong Kong researcher Dennis Lo – who's own fetal DNA sequencing method underlies another commercial non-invasive prenatal aneuploidy test marketed by Sequenom – demonstrated last month in a study in Clinical Chemistry the feasibility of using RNA-seq to profile fetal and maternal RNA transcripts in maternal blood.