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Stanford-Led Team Develops Sequencing-Based Maternal Blood Test for Fetal Chromosomal Abnormalities

NEW YORK (GenomeWeb News) – Using shotgun sequencing, researchers from Stanford University and the Howard Hughes Medical Institute have developed a way to test for fetal chromosomal abnormalities, including Down syndrome, based on a mother’s blood sample.
In a paper appearing online last night in the Proceedings of the National Academy of Sciences, Stanford University bioengineer Stephen Quake and his team demonstrated that their approach was effective in a small study of 18 women. If those results hold in larger studies, researchers say, the test could eventually enter the clinic — replacing more invasive testing procedures such as amniocentesis and chorionic villus sampling.
“We’re using the fruits of the human genome project,” Quake told GenomeWeb Daily News. By shotgun sequencing the mixture of fetal and maternal DNA in maternal blood and mapping the reads back to the genome, the team can see which chromosomes, if any, are over- or under-represented.
Fetal aneuploidy and other types of chromosomal aberrations affect roughly nine in every thousand live births. At the moment, women who are candidates for prenatal genetic testing typically undergo amniocentesis, sampling the amniotic fluid, or chorionic villus sampling, testing a small piece of the placenta. But both of these approaches require doctors to insert a needle in the uterus — a procedure that increases the risk of miscarriage by about a half a percent.
“Right now, people are risking their pregnancies to get this information,” co-author Yair Blumenfeld, a post-doctoral researcher in obstetrics and gynecology at Stanford, said in a statement.
In the past, Quake explained, others have tried to come up with methods for measuring the mixture of fetal and maternal DNA in maternal blood. But he noted that so far those attempts have come up with population-based screens rather than methods for actually measuring fetal chromosome content.
In an effort to remedy that, Quake and his team looked at whether they could use digital PCR to amplify fetal DNA from maternal blood samples.
“We and others argued that it should be possible, in principle, to use digital PCR to create a universal, polymorphism-independent test for fetal aneuploidy by using maternal plasma DNA,” Quake and his colleagues wrote, “but because of technical challenges relating to the low fraction of fetal DNA, such a test has not yet been practically realized.”
Instead of trying to isolate fetal DNA and then sequence it, the team used shotgun sequencing to sequence all of the cell-free DNA in the mother’s blood plasma. For this paper they mainly used an Illumina Genome Analyzer to randomly sequence a mixture of maternal and fetal DNA fragments that were between 25 and 30 base pairs long.
They then determined how many sequences came from each chromosome to determine how many copies of each chromosome were present. For instance, a woman carrying a fetus with Down syndrome, or trisomy 21, will have more chromosome 21 sequences in her blood.
“By counting the number of sequence tags mapped to each chromosome, the over- and under-representation of any chromosome in maternal plasma DNA contributed by an aneuploid fetus can be detected,” the authors wrote.
Not every chromosome was equally represented, though. To adjust for the non-uniform distribution of sequence tags from one chromosome to the next, the researchers applied a 50,000 base “sliding window” to come up with normalization values for each, minimizing the effects of noise from sequence bias.
For the 18 pregnancies tested, the researchers detected all nine cases of Down syndrome as well as two cases of trisomy 18 (also known as Edward syndrome), and a lone case of trisomy 13, called Patau syndrome. These chromosomal abnormalities were evident in a mother’s blood as early as 12 or 14 weeks into her pregnancy.
In theory, Quake said, the approach should be useful for picking up all potential chromosomal aberrations. But because the main sequencing method used in the paper has a slight G/C bias, he said, it may not be possible to get at absolutely all potential chromosomal aberrations just yet.
Quake said the team also plans to test the method using other sequencing technologies as well, including the Helicos BioSciences platform, which relies on a single-molecule sequencing approach that Quake helped to pioneer.
Quake, who is also a founder of and consultant for Fluidigm, has applied for a patent related to the methodology outlined in the paper, along with lead author Christina Fan, a bioengineering graduate student at Stanford University. Quake could not disclose who potential licensees of the technology might be.
The current cost of the assay is about $700. Quake predicts the cost will drop to roughly $300 as sequencing costs decrease. If larger trials — currently in the design stage — prove successful, Quake estimated that the blood test could reach the clinic in two or three years.
“My goal is to make invasive technologies obsolete,” he said.
San Diego-based firm Sequenom is currently running clinical trials on its own non-invasive test for Down syndrome. It recently announced that the SEQureDx test successfully tested for Trisomy 21 in 219 patients with no false positives or negatives.

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