Researchers from Maastricht University in the Netherlands this week presented early-stage results for an inexpensive prenatal blood test to detect chromosomal abnormalities such as trisomy 21.
The test, which is based on MRC-Holland's Multiplex Ligation-dependent Probe Amplification technology, interrogates cell-free fetal DNA extracted from maternal blood plasma drawn in the first trimester.
This week at the annual meeting of the European Society of Human Reproduction and Embryology in Rome, Suzanna Frints, a clinical geneticist at Maastricht University Medical Center, presented proof of principle that the test could identify DNA from the Y chromosome in male fetuses in maternal blood.
The researchers are further developing the method to detect trisomy 21, which causes Down syndrome, and will then move on to trisomy 13 and 18, which are responsible for Patau and Edward’s syndromes, respectively.
The study began in 2009 and its researchers expect to make the test available for clinical use within five years.
In the presentation, which was webcast, Frints noted that noninvasive techniques such as ultrasound can flag fetuses at elevated risk for Down syndrome, but the only reliable confirmatory methods are invasive procedures such as amniocentesis or chorionic villus sampling. Both techniques can trigger a miscarriage.
The aim of the Maastricht test is to be simple, inexpensive, fast, and reliable, she said. The team predicts that it will be able to deliver all of these characteristics with a one-tube test that can generate results in one to three days with 100 percent specificity for under $100.
In a preliminary study of 30 women, results obtained with the MLPA test were compared with the results of amniocentesis, chorionic villus sampling, and pregnancy outcome.
"All but one sample correlated with the non-invasive MLPA test results, detecting fetal Y-chromosome sequences,” Frints said.
She estimated that the reliability of the test is currently around 80 percent due to false negative results, but added that she and her colleagues are "working to improve the accuracy of the MLPA probe."
The MLPA test is part of a kit that is already widely used to detect chromosomal abnormalities in amniotic fluid or chorionic villi samples. Until the Maastricht study it was not known whether it would work on cell-free fetal DNA circulating in maternal blood.
The Dutch company MRC-Holland has developed some probes for the project, but hasn't had much involvement beyond that. Company CEO Jan Schouten, told PCR Insider that MLPA typically requires a minimum of 20 ng of DNA, but fetal DNA is found in very low concentrations in maternal blood, making it difficult to obtain the optimal quantity.
However, he said, the Maastricht team likely preamplified the sequences detected by the MLPA probes or used an extra enrichment step in order to reduce the minimum amount required.
Schouten said that MLPA offers a number of advantages over other approaches for detecting copy number changes. For example, he said it is more accurate than real-time PCR in detecting small copy-number changes and can be multiplexed. It also offers cost and performance advantages over fluorescent in situ hybridization and microarray-based tests in certain applications, he said.
A Test in Five Years?
Joep Geraedts, head of the Department of Genetics and Cell Biology at Maastricht University, told PCR Insider that the team recently received a grant of an undisclosed amount from the Netherlands government to modify the approach for detecting trisomies, which is a "very different" task than identifying the Y chromosome.
"If you have a Y chromosome, you can detect it and say there is a male baby, and if there is no Y chromosome you know the baby is female, but with trisomy 21 it's different because everyone has got two copies of chromosome 21, so what you want to do is see the difference between three copies of the chromosome and two copies," he said. "It's not a qualitative difference, but a quantitative difference, and that makes it more difficult."
Geraedts estimated that it will take "a couple of years" to perfect the test for detecting trisomies. The study, which began in 2009 and is expected to run until 2012, is expected to yield a marketable clinical test by 2015.
For a follow-on study, the researchers are currently recruiting women who are at high risk of an abnormal pregnancy and undergoing prenatal screening and invasive diagnostic procedures. The goal is to recruit approximately 800 women across four different subgroups in order to test the reliability of the MLPA technique.
The Maastrict team joins a number of other groups developing noninvasive prenatal diagnostics that interrogate fetal DNA taken from maternal blood. For example, researchers from Stanford University published a paper two years ago in the Proceedings of the National Academy of Sciences that described the use of high-throughput sequencing to detect trace amounts of fetal DNA in a pregnant woman's blood to determine whether any chromosomes are over-represented. That technology has been licensed to Fluidigm and Artemis Health.
Other companies, such as Sequenom and LifeCodexx, are developing similar noninvasive tests that would run on next-generation sequencing instruments.
Geraeds acknowledged that "you can do more" with sequencing than with MLPA, "but the difference is that it is more time consuming, and it is much more expensive — at the moment, at least."
In addition, the sheer amount of data produced by sequencing can pose its own problems in the prenatal diagnostic setting.
"With our technique, [a chromosomal abnormality is] either there or it isn't, but with sequencing you always come across abnormalities, and then the interpretation of these is the difficult part: Is it pointing towards something that is severe, or is it mild?" he said. "Or is it new, in which case you have to [sequence] the parents and compare what you find to the baby, and so on. So it makes it much more complicated, and we're aiming for a much more simple method."