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Sequenom, Collaborators Demonstrate Efficacy of Non-Invasive Prenatal Monogenic Disease Test

This article has been updated with a comment from Sequenom regarding its rights to the technology.
NEW YORK (GenomeWeb News) – Researchers from Sequenom, the Chinese University of Hong Kong, and Thailand’s Chiang Mai University have developed a prenatal PCR-based testing approach for detecting single gene mutations in fetal DNA found in maternal blood samples.
In a paper scheduled to appear online this week in the Proceedings of the National Academy of Sciences, the team demonstrated that its “mutation counting” approach can detect mutated fetal sequences in maternal blood — particularly when it used a size-selection strategy to enrich for fetal DNA. Eventually, the researchers said, such an approach could reduce the use of invasive chorionic villus and amniocentesis in monogenic disease testing.
“This research represents a significant paradigm shift in the way we approach plasma DNA-based diagnostics, and offers substantial promise for bringing non-invasive prenatal diagnosis of monogenic diseases closer to reality,” senior author Dennis Lo and Li Ka Shing, researchers at the Chinese University of Hong Kong, said in a statement.
More than a decade ago, Lo was first author on a study demonstrating the ability to detect fetal DNA in maternal blood. Subsequent research suggests it’s possible to test this DNA for fetal sex-linked disorders, RhD status, and trisomy 21. But detecting single gene mutations — such as those responsible for cystic fibrosis or the inherited anemias β-thalassemia and hemoglobin E — has proven more difficult.
At present, tests for these and other genetic conditions generally rely on invasive procedures such as chorionic villus sampling and amniocenteses, which are associated with a small miscarriage risk.
In an effort to detect these types of mutations in fetal DNA in a mother’s blood, the researchers used an approach called relative mutation dosage, or RMD, comparing the relative amounts of mutated and normal DNA sequence present in each sample.
“Digital PCR technologies have enabled us to measure the minute imbalance of mutant and normal DNA sequences in maternal plasma, due, for example, to contribution by a fetus that has inherited two doses of the disease gene,” Lo and Shing explained.
To develop their approach, the team did a series of RMD experiments using Fluidigm’s Biomark System and its 12.765 Digital Arrays.
First, they obtained blood samples from a dozen male and a dozen female subjects, mixing the male and female samples to create 12 mixtures containing three times as much male DNA. By targeting the X-linked gene ZFX and the chromosome 7 gene ZFY, the researchers showed that they could correctly classify the female DNA in each mixture.
The researchers also validated their test for identifying the mutations behind β-thalassemia and hemoglobin E and then tested it on a group of pregnant women carrying male fetuses.
For instance, when the team used the RMD approach to test blood samples from ten pregnant women in their second trimester who carried the β-thalassemia (CD41/42) mutation or the hemoglobin E (HbE), mutation, the researchers correctly classified fetal genotypes about half of the time. One case was misclassified and four samples remained unclassified (though these had fetal DNA concentrations of less than ten percent).
A computer simulation suggested that, in order to group the unclassified cases, the researchers would have to either use larger maternal blood samples or enrich the amount of fetal DNA.
“The diagnostic performance of digital RMD is dependent on an interplay between the fractional fetal DNA concentration and number of DNA molecules in maternal plasma,” the authors wrote.
Because cell-free fetal DNA found in maternal blood is typically shorter than maternal DNA, the team used size selection to enrich fetal DNA in the blood samples. They applied digital nucleic acid size selection, or NASS, using duplex digital PCR to generate long and short amplicons that are identified by mass spectrometry.
“The advantage of NASS is that it allows locus-specific enrichment of fetal DNA without the need for extra sampling of maternal plasma or additional experimental time,” the authors explained.
When the team tested NASS assays designed to detect ZFX and ZFY in ten first-trimester women carrying male fetuses, the researchers found that they could enrich the amount of fetal DNA sampled by 38 percent. Likewise, when they tested a digital NASS assay designed to assess a SNP in the PLAC4 gene, fetal DNA was enriched by nearly 33 percent.
Accordingly, by combining the NASS assay with RMD analysis, the researchers improved the number of cases in which they could accurately detect mutations of interest. For example, when they tested five pregnant women who were heterozygous for the PLAC4 SNP and carrying male fetuses, the team correctly classified four cases without applying the PLAC4 NASS. But with the assay, they classified all five case correctly.
Subsequent computer simulations of 5,000 cases suggested that NASS assays would classify ten percent of heterozygous and homozygous cases beyond the 75 percent or so correctly classified by the RMD technique alone.
Although the approach was only tested with a limited set of mutations in male fetuses for this paper, the authors suggest it can likely detect monogenic mutations behind a range of autosomal dominant and autosomal recessive diseases in fetuses of both sexes.
“With the gradual dismantling of the obstacles to non-invasive prenatal diagnosis, clinical trials could be set up to investigate the clinical performance of these new tests,” Lo and his team concluded.
Other than the use of the Fluidigm products, Sequenom has exclusive rights to the technology described in the new paper. "Everything is proprietary, including the use of digital PCR for prenatal diagnostics," according to a Sequenom official.
The company also is developing a non-invasive prenatal test for Down syndrome and other genetic conditions. Sequenom has published data demonstrating a high level of accuracy for its test. However, it could face some competition from a new method developed Stephen Quake and his colleagues at Stanford University.
Last month, researchers from Stanford and elsewhere reported that they could successfully detect fetal Down syndrome in maternal blood using a shotgun-sequencing-based test.
In late Tuesday trade on the Nasdaq, Sequenom's shares were up around 4 percent at $15.46.

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