Chromosomal aneuploidy is the major reason why couples opt for prenatal diagnosis. Current methods for definitive diagnosis rely on invasive procedures, such as chorionic villus sampling and amniocentesis, and are associated with a risk of fetal miscarriage. Fetal DNA has been found in maternal plasma but exists as a minor fraction among a high background of maternal DNA. Hence, quantitative perturbations caused by an aneuploid chromosome in the fetal genome to the overall representation of sequences from that chromosome in maternal plasma would be small. Even with highly precise single molecule counting methods such as digital PCR, a large number of DNA molecules and hence maternal plasma volume would need to be analyzed to achieve the necessary analytical precision. Here we reasoned that instead of using approaches that target specific gene loci, the use of a locus-independent method would greatly increase the number of target molecules from the aneuploid chromosome that could be analyzed within the same fixed volume of plasma. Hence, we used massively parallel genomic sequencing to quantify maternal plasma DNA sequences for the noninvasive prenatal detection of fetal trisomy 21. Twenty-eight first and second trimester maternal plasma samples were tested. All 14 trisomy 21 fetuses and 14 euploid fetuses were correctly identified. Massively parallel plasma DNA sequencing represents a new approach that is potentially applicable to all pregnancies for the noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.Down syndrome ͉ Solexa sequencing ͉ trisomy 21

The discovery of circulating fetal nucleic acid in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. Thus far, a gender-and polymorphism-independent fetalspecific target that can be used for prenatal screening and monitoring in all pregnant women has not been reported. In addition, the origin of such circulating nucleic acid has remained unclear. Here we provide direct evidence that the placenta is an important source of fetal nucleic acid release into maternal plasma by demonstrating that mRNA transcripts from placenta-expressed genes are readily detectable in maternal plasma. The surprising stability of such placental mRNA species in maternal plasma and their rapid clearance after delivery demonstrate that such circulating mRNA molecules are practical markers for clinical use. The measurement of such plasma mRNA markers has provided a gender-independent approach for noninvasive prenatal gene expression profiling and has opened up numerous research and diagnostic possibilities.N oninvasive prenatal diagnosis is a long-sought goal in human genetics. Recent interest in cell-free DNA in plasma and serum (1, 2) has led to the discovery of fetal DNA in maternal plasma (3)(4)(5). This noninvasive source of fetal nucleic acid has already been shown to be clinically valuable in the prenatal investigation of many conditions, including fetal rhesus D status (6, 7), sex-linked diseases (8), and ␤ thalassemia (9). In addition, quantitative aberrations of fetal DNA have been described in many pathological conditions, including preeclampsia (10, 11), fetal chromosomal aneuploidies (12, 13), and hyperemesis gravidarum (14).Despite the promising clinical use of fetal DNA in maternal plasma for noninvasive prenatal diagnosis, a number of challenges remain and several fundamental biological issues about this phenomenon are unresolved. First, in studies reporting the quantitative abnormalities involving fetal DNA in maternal plasma, the Y chromosome is commonly used as a fetal-specific marker in women carrying male fetuses (10-14). The use of such Y-specific markers has limited the application of this technology to the 50% of pregnant women who are carrying male fetuses. The eventual routine clinical application of this technology, e.g., as a screening tool for fetal chromosomal aneuploidies (12, 13), will be catalyzed by the development of a gender-and polymorphism-independent fetal nucleic acid marker, which can be used in all pregnancies. Second, the source of fetal DNA in maternal plasma remains unclear. Although it has been suggested that such fetal DNA could have originated from the placenta (4), no empirical proof of this hypothesis has been put forward to date.Recently, a number of investigators have shown that in addition to DNA, RNA is also present in the plasma of human subjects, particularly those with cancer (15-18). The inherent lability of RNA has made these observations rather surprising. It has been suggested that circulating RNA may be stabilized by being protected in apoptotic ...

Trisomy 21 is the most common reason that women opt for prenatal diagnosis. Conventional prenatal diagnostic methods involve the sampling of fetal materials by invasive procedures such as amniocentesis. Screening by ultrasonography and biochemical markers have been used to risk-stratify pregnant women before definitive invasive diagnostic procedures. However, these screening methods generally target epiphenomena, such as nuchal translucency, associated with trisomy 21. It would be ideal if noninvasive genetic methods were available for the direct detection of the core pathology of trisomy 21. Here we outline an approach using digital PCR for the noninvasive detection of fetal trisomy 21 by analysis of fetal nucleic acids in maternal plasma. First, we demonstrate the use of digital PCR to determine the allelic imbalance of a SNP on PLAC4 mRNA, a placenta-expressed transcript on chromosome 21, in the maternal plasma of women bearing trisomy 21 fetuses. We named this the digital RNA SNP strategy. Second, we developed a nonpolymorphism-based method for the noninvasive prenatal detection of trisomy 21. We named this the digital relative chromosome dosage (RCD) method. Digital RCD involves the direct assessment of whether the total copy number of chromosome 21 in a sample containing fetal DNA is overrepresented with respect to a reference chromosome. Even without elaborate instrumentation, digital RCD allows the detection of trisomy 21 in samples containing 25% fetal DNA. We applied the sequential probability ratio test to interpret the digital PCR data. Computer simulation and empirical validation confirmed the high accuracy of the disease classification algorithm.circulating fetal nucleic acids ͉ noninvasive prenatal diagnosis ͉ sequential probability ratio test ͉ trisomy 21 ͉ RNA SNP

Background: Circulating RNA in plasma/serum is an emerging field for noninvasive molecular diagnosis. Because RNA is widely thought to be labile in the circulation, we investigated the stability and various preanalytical factors that may affect RNA concentrations in blood specimens. Methods: Blood samples were collected from 65 healthy volunteers. The effects of two preanalytical variables were studied: (a) time delay in processing of EDTA blood and clotted blood after venesection, and (b) freezing and thawing of plasma and serum. The lability of free added RNA in plasma was also investigated. Plasma/serum RNA was measured by a real-time quantitative reverse transcription-PCR assay for glyceraldehyde 3-phosphate dehydrogenase mRNA, whereas DNA was measured by a real-time quantitative PCR assay for the β-globin gene. Results: No significant difference was found for plasma RNA concentrations obtained from uncentrifuged EDTA blood that had been left at 4 °C for 0, 6, and 24 h (P =0.182). On the other hand, the serum RNA concentrations increased significantly over 24 h when uncentrifuged clotted blood was stored at 4 °C (P <0.05). In comparison, >99% of the free added RNA could no longer be amplified after incubation in plasma for 15 s. Never-frozen plasma, freeze-thawed plasma, and thawed plasma left at room temperature for 1 h showed no significant differences in RNA concentration (P =0.465). No significant difference was observed for freeze-thawed serum (P = 0.430). Conclusions: Plasma RNA is stable in uncentrifuged EDTA blood stored at 4 °C, but to obtain a stable serum RNA concentration, uncentrifuged clotted blood should be stored at 4 °C and processed within 6 h. A single freeze/thaw cycle produces no significant effect on the RNA concentration of plasma or serum.