Helen Albert Writer GEN

Prenatal Genetic Testing Looks to Single-Cell Methods as the Next Frontier

Prenatal genetic testing has advanced enormously over the last few decades. Amniocentesis and chorionic villus sampling (CVS) have been performed to diagnose fetal abnormalities for many years, but these tests are invasive and not without problems. The risk for miscarriage is low (approximately 1 in 1000 for amniocentesis and 1 in 500 for CVS), but it has prompted a long quest to find the perfect noninvasive prenatal test.

The recent advent of fetal cell-free DNA (cfDNA) tests has significantly changed the prenatal testing landscape. “We published the first large-scale trial on the non-invasive Down syndrome tests in January 2011 and within 10 months the test was launched in the USA,” prenatal cfDNA test founder Dennis Lo, M.D., Ph.D., Chinese University of Hong Kong, told Clinical OMICs. “The test is now used in over 90 countries and millions of tests are done every year.”

With a greater accuracy than standard noninvasive screening (maternal serum testing for protein markers and ultrasound scanning) for predicting risk of more common genetic disorders such as Down (trisomy 21), Patau (trisomy 13), and Edwards (trisomy 18) syndromes, cfDNA testing has become a popular option for patients and providers.

While these tests have improved the accuracy of early screening for more common aneuploidies, they currently only have limited accuracy for detecting rarer conditions such as DiGeorge syndrome (22q11 deletion). They also still require an invasive diagnostic test to definitively confirm the result, as false positives do occur.

The presence of fetal cells in maternal blood was first detected in 1969. Since then, extracting these cells for analysis has become the “Holy Grail” of prenatal testing with many researchers and companies trying and failing to successfully extract and analyze these cells. In 2016, a number of papers were published demonstrating moderate success in this area, and what has in the past seemed an unachievable goal has moved slightly closer to reality.  


Cell-Free DNA Revolution

In the late 1990s, Dr. Lo and colleagues published research showing that cfDNA could be detected in maternal blood and that a significant proportion, made up of placental trophoblast cells, was from the fetus.

The first maternal blood tests for fetal cfDNA on the market were for Down syndrome alone, but very quickly went on to cover Patau and Edwards syndromes and sex chromosome aneuploidies. Six years down the line, tests for many additional abnormalities, including deletion syndromes such as DiGeorge, have been developed, but the accuracy at detecting these sub-chromosomal abnormalities remains suboptimal.

“There are cfDNA-based diagnostic tests targeting known microdeletions but more data is needed to establish performance characteristics in large high-risk and low-risk populations,” commented molecular diagnostics expert Ahmad Abou Tayoun, Ph.D., from the University of Pennsylvania.

Dr. Tayoun added: “Although fetal sequence variant detection can be, and has been, performed using cfDNA on a research basis, this is still not mature enough to be used routinely in the clinic. Such testing requires sophisticated yet highly reproducible and robust bioinformatics pipelines (to detect the smaller proportion of fetal DNA [~10%] in maternal blood) that are still beyond the capabilities of most molecular diagnostic labs.”

When asked about performance of cfDNA tests for subchromosomal disorders, Dr. Lo said: “There is actually no consensus at the moment.” But he added that “a number of experts in the field are trying to improve the specificity of the tests by looking at characteristics of the fetal DNA that can distinguish it from the maternal DNA in the mother’s blood.”

Dr. Lo’s team has shown that fetal cfDNA is shorter than maternal cfDNA. They have also discovered that when the DNA breaks into fragments in plasma, there are some preferred ending sites that could be preferentially cleaved if the DNA is from the mother versus from the baby and vice versa.

“A key factor that will enhance the use of cfDNA clinically, is innovative ways to enrich or isolate the fetal DNA from maternal circulation and then perform sequencing,” commented Dr. Abou Tayoun.

Currently, cfDNA sequencing has to be performed at a much higher depth than sequencing performed on CVS or amniocentesis samples, he explained. Yet, despite the higher depth of sequencing, the chance of missing a variant is still quite high compared with standard diagnostic testing samples. 


Quest for Single-Cell Analysis

Clinician and molecular diagnostics expert Arthur Beaudet, M.D., Baylor College of Medicine, has been working to develop cell-based prenatal screening for many years and has recently had success with cell-based testing in a research setting. He published two papers last year, one with U.S.-based single-cell analysis company Rarecyte, and a second with Danish firm Arcedi Biotech, both of which demonstrated the feasibility of a screening technique targeting fetal trophoblast cells.

He explained that Rarecyte uses a technique that microscopically scans all the nucleated cells in the sample and identifies ones that stain positively for a protein called cytokeratin, whereas Arcedi has taken a different approach of trying to identify antibodies that will bind specifically to the fetal cells and then use magnetic bead try to pull out fetal cells from the maternal cells.

In his current work, Dr. Beaudet and his team are using antibodies to enrich for the fetal cells and then picking individual cells after cytokeratin staining.

“The downstream part, to take the cell and analyze its DNA, is done quite a lot in preimplantation genetic screening and preimplantation genetic diagnosis. So the techniques for the downstream part are relatively well established, but it’s getting hold of the cells that is the challenge,” said Dr. Beaudet.

He added that finding ways to accurately scale up the cell-based testing methods are needed. “I would love to be able to launch a test even at low volume very, very soon, but I just don’t know. It’s tricky to make guesses,” he commented.   

Ripudaman Singh, Ph.D., chief technical officer at Arcedi Biotech, highlighted why analyzing whole fetal cells is so advantageous. “Because cell-based non-invasive prenatal diagnosis technologies offer intact fetal cells with access to the complete fetal genome, uncontaminated with maternal DNA, the possibilities of even detecting small changes (eg., copy number variations) in the fetal genome are immense.”

In addition to the three most common aneuplodies, Arcedi Biotech has detected subchromosal abnormalities, such as a 31–Mb translocation between chromosomes 4 and 8 and a 12.2–Mb duplication on chromosome 21, with their test. These results were confirmed using CVS.

Dr. Singh said that the company plans to roll out its cell-based noninvasive prenatal diagnosis tests to six different regional hospitals in Denmark later this year.

“This will the first time since 1969, when using fetal cells for chromosomal aneuploidy detection was first proposed, that pregnant women will get an opportunity to opt for cell-based noninvasive prenatal diagnosis in a clinic,” he said.

Although it is a smaller field than that of cfDNA testing, a number of other groups are developing cell-based prenatal tests. Shuang Hou, Ph.D., a researcher at UCLA is working on enriching circulating fetal trophoblast cells using “NanoVelcro Microchips” and then isolating them using laser capture microdissection.

Similar to Dr. Beaudet, he says that while the technique is working reasonably accurately in the lab, scaling it up to use in the clinic is the next step. “Our plan is to push it to the market in 2 or 3 years to make sure that people can use it, I’m not sure what kind of technical problems we are going to encounter, but that’s our plan.”

Researchers from Wayne State University School of Medicine also published work last year showing they could profile fetal trophoblast cells extracted from the endocervical canal (in a technique similar to that used in the Pap smear test) at five weeks’ gestation.


Future Directions

In the coming years, it seems likely that use of cfDNA testing will expand and also become more accurate for rare conditions. It also seems probable that at least one of the teams developing cell-based tests will have achieved a “clinic-ready” test within the next couple of years. 

Dr. Lo believes that taking into account the various recently discovered characteristics of fetal DNA, such as the size of the fragments, specific cutpoints, and methylation status (fetal DNA is generally hypomethylated) can help develop more accurate cfDNA tests for a wider range of disorders.

One somewhat neglected area he hopes will expand is testing for monogenic disorders, such as congenital adrenal hyperplasia, using cfDNA.

He thinks one reason these tests have not been widely commercialized could be because assays would ideally need to be tailored to specific groups or families, but says that this shouldn’t be an insurmountable issue if a company really wished to develop this further.

In a bid to make cfDNA tests more accessible in low-resource settings, European noninvasive prenatal testing company LifeCodexx recently created a cfDNA platform using qPCR. “This technology can provide the basis for an affordable, cost-efficient noninvasive prenatal testing solution that could be deployed with minimal investment around the globe in a local and decentralized manner,” CEO Michael Lutz, Ph.D., explained.

Notably, the company’s more expensive PrenaTest currently tests for DiGeorge syndrome in addition to more common abnormalities. When asked about how accurately the company’s test is at detecting the deletion, Dr. Lutz commented: “Since DiGeorge syndrome was introduced about oneyear ago, we have not experienced any false-positive or false-negative results so far.”

When considering the future of noninvasive prenatal testing, Dr. Beaudet commented: “I would like to see a next phase in which we try to sequence the entire fetal genome or certain parts of the fetal genome to find de novo point mutations, which can be very deleterious and altogether account for maybe five times as much disability as Down syndrome.

 “Our lab has launched a test that’s beginning to look for these mutations, which are highly correlated with the age of the father,” he added.

Dr. Singh believes that “a simpler, safer, and accurate prenatal diagnosis based on fetal cells is the future.”

In a challenge to proponents of cfDNA testing, he commented: “In the near future, we see cell-based noninvasive prenatal diagnosis taking over from cell-free noninvasive prenatal testing. In the long run, it will replace invasive prenatal diagnostics methods.”







































This article was originally published in the July/August 2017 issue of Clinical OMICs. For more content like this and details on how to get a free subscription to this digital publication, go to www.clinicalomics.com.

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