Detecting Genetic Disease Prior to Birth

The advent of noninvasive prenatal testing allow for the use of a simple blood draw from a pregnant woman to tests fetal DNA for genetic conditions. As with liquid biopsies, these test rely on capturing cell-free DNA from the fetus circulating in the mother’s blood. We spoke to Paul Billings, chief medical officer for Natera, about the state of non-invasive prenatal testing, the growing use of these tests, and the range of conditions they can detect.

Daniel Levine: Paul, thanks for joining us.

Paul Billings: Happy to be with you.

Daniel Levine: We’re going to talk about cell-free DNA, Natera, and how this is being used to diagnose rare disease prior to birth. Perhaps we can start with cell-free DNA itself. This is a term that people may be familiar with in the context of liquid biopsies. What is cell-free DNA?

Paul Billings: Cell-free DNA is DNA, which is present in virtually every cell of the body and is released by those cells. That release can occur for a variety of metabolic processes. That can be something called apoptosis, which is a programmed cell death. It can be because that cell was attacked by other cells and can be or injured in some way—that can be necrosis. There are several other pathways that release cell-free DNA, maybe some of that DNA is a signal to other cells, but in any event, it’s fragments of DNA, which are released and are measured in a fluid, usually the blood but it also could be the urine or the cerebral spinal fluid, or maybe even in the bowels or the saliva. So, it’s a fragment of the genetic material that is released and is assayable in fluids of the body.

Daniel Levine: And the case of women who are pregnant, is fetal DNA always present in the blood in some form?

Paul Billings: The fetus and the mother are in intimate blood connection. The placenta and other touch points where the fetus is growing inside the womb interplay with the maternal circulation. And so to the extent that the fetus has a circulation, it is shared with the mother prior to birth. So yes, the fetus makes cell-free DNA and yes, the mother makes cell-free DNA. So, during the nine months of gestation fetal and maternal cell-free DNA are in a mixture and can be assayed, potentially in the fetal blood, but that’s obviously hard to get at so you use the maternal blood.

Daniel Levine: I suspect that in terms of quantity, maternal blood overwhelmingly contains maternal DNA. How detectable is fetal DNA in maternal blood, and how do you determine whether you’re reading maternal DNA or fetal DNA?

Paul Billings: So, fetal DNA, as the fetus grows older, becomes a significant component of the maternal circulation, and frankly is much easier to detect when the fetus is at 20 or 30 weeks of gestation than when it’s at 10 weeks of gestation. And it can be very difficult to detect let’s say before nine weeks of gestation. Now there are different methods by which one can detect the fetal component of the maternal cell-free DNA population. Different methods have different sensitivities and specificities and different failure modes. In other words, some methods are so complex that to actually get to a reading has been historically problematic, but with developments in a highly multiplex polymerase chain reaction of the so-called Xerox machine of DNA, with advancements in that world, as well as in advancements in the ability to make libraries and do sample development for cell-free DNA assays, as well as the bioinformatics on the backend where you differentiate fetal DNA from maternal DNA, from the noise of these assays—all these advancements have allowed certain methods to be exquisitely sensitive and exquisitely effective at detecting the cell-free DNA from the fetus.

Daniel Levine: So being able to detect and read this fetal DNA has allowed for non-invasive prenatal testing, which is known as NIPT. This offers the potential to detect genetic disease prior to birth. How many different conditions are screened with this type of test today?

Paul Billings: Well again, it varies a little bit about which test you apply. Many of the providers of NIPTs use simply a chromosome counting methodology, and that can be applied and that has a sensitivity for certain kinds of chromosomal arrangements. Our method uses thousands of polymorphisms—changes in the DNA that differentiate the fetal DNA from the maternal DNA. And that method is particularly robust at detecting the common trisomies. So that’s trisomy 21, trisomy 18, trisomy 13, but other key differences as well, like a monosomy X, like a whole extra compliment of chromosomes called triploidy. Uh, the sex chromosomes also have a pretty high frequency of variation. Monosomy X is one of them, but also XXX, XXY, XYY, those are common polymorphisms of the sex chromosomes. Those are very important. And then there’s the whole world of the so-called microdeletion syndromes. These are well-described newborn conditions that involve small either additions or deletions of pieces of DNA at specific sites within the genome. Uh, the most common one that we often talk about is the 22q deletion syndrome, but there are things like Prader-Willi and Cri-du-chat, and Angelman syndrome. These are all areas where our technology can easily detect these kinds of aberrations in the fetus with a great deal of sensitivity. We also have a separate kind of analysis when there is a suspicion that there is a monogenic, usually dominant de novo disorder. You know, let’s say that a mother has had a pregnancy that’s had an unexpected finding of osteogenesis imperfecta, or a form of dwarfism like achondroplasia in a previous pregnancy. So that pregnancy is at high risk for a second pregnancy of that sort. Or let’s say that there’s been an ultrasound done, and that ultrasound is suggestive of some monogenic new disorder in that fetus. We can do our Vistara assay, which looks at 25 rare genetic syndromes like Noonan’s syndrome, like achondroplasia, like osteogenesis imperfecta, and we can assess that pregnancy specifically for the presence of a fetus with those anomalies. And that test is incredibly specific and sensitive to the detection of those disorders. So, we have the Panorama assay, which is our highly multiplex PCR that we’ve modified and it’s gotten better and better. That detects the trisomies, the sex chromosome abnormalities, the microdeletion syndromes, and by the way, is very good at distinguishing monozygotic from dizygotic, twin pregnancies. And that’s a very important distinction as well because, monozygotic pregnancies have a set of complications that di-zygotics generally do not. So, you can manage dizygotic pregnancies differently as an OB or a maternal fetal medicine specialist than you would if you were managing a monozygotic pregnancy. And then we have this other side of the house, which is high-risk pregnancies that we’re worried about because of other findings where we might apply the Vistara assay.

Daniel Levine: My sense, is from a technology point of view we could detect a far greater number of conditions with this type of testing than we’re using today. What determines what conditions would be tested for?

Paul Billings: Well, there really are two sides of the coin there. One is what the unmet need is and what we could do something about, right? I mean the pressure to add things is to add things that make a difference to that mother as she carries that fetus, to the fetus so that it develops as normal as it can while it’s still in utero, and then for its flourishing after birth. So, to the extent that there are proven interventions or proven drugs that can make a difference at having identified something that’s potentially variant within the pregnancy, that’s one major criteria. The other criteria, of course, is access to a fetal assessment, whether it be cell-free DNA in the maternal circulation or other kinds of fetal determination, whether it be through amniocentesis or through other methods like ultrasound and so forth and using that information and mining that information for inferences about the condition of the fetus. So, it’s getting the most information you can from the biomarkers and analytes that you have available to you. And then it’s also marrying that with clinical utility and clinical effectiveness that you can do something with that information, either manage that pregnancy with more interventions, manage that pregnancy with less interventions, and deliver that child in a setting that optimizes its health later on.

Daniel Levine: What’s the case for using this testing today? What benefits does it provide for the conditions that you’re able to detect?

Paul Billings: Well, let’s take an example. Many people are familiar with trisomy 21 or Down syndrome, as it was historically called, because there are many children that are born with that disorder. There are many children who are stillborn or have a miscarriage, many pregnancies and miscarriages with that variation, but a significant number of them are born and people have a familiarity with that. Well, it’s very evident that those kids have cardiac and other kinds of skeletal problems as they develop in utero. They can have metabolic issues at birth or near birth and other kinds of developmental issues, neurodevelopmental issues, which can be modified. So, the earlier you identify a pregnancy with down syndrome the more you can provide interventions either in utero or at birth or around birth that improve the outcome of those pregnancies. That’s a major finding. That’s true, by the way, of several of the microdeletion syndromes. There’s quite good evidence that identifying the microdeletion syndromes in utero can improve the later stage fetal development and then the newborn care of those individuals. Those pregnancies can be, instead of delivered by a midwife in a rural situation, delivered at a tertiary care center that’s perfectly capable of taking care of the complex management needs of some of those children. So, those are, I think, good examples where this kind of thing can happen. And that’s true by the way of the Vistara disorders as well—the 25 rather rare monogenic disorders. Many of them have a collagen abnormality and if you try to deliver those in certain situations, you can really harm the child as it’s being delivered. So, you need to really be in a unique setting to deliver those kids.

Daniel Levine: Well, walk me through how the test works. Take me from getting a sample from a patient to the result.

Paul Billings: Well, so we’re talking about both the Panorama test, which is our noninvasive prenatal test that we offer for all pregnancies, or the Vistara test, which is increasingly being applied for pregnancies that are known to be at high risk or other pregnancies where the information about this cadre of 25 rare disorders is desired. Basically, as early as nine or 10 weeks we take a blood sample. The blood sample is 10 to 20 CCs and is transported to our lab where the sample is prepped. And the DNA is isolated from that sample and that DNA, as I said, is a mixture of maternal and fetal DNA. Then the particular method that we apply, the highly multiplex PCR method that we apply is then done in the laboratory. That takes maybe a week with the sample prep and the DNA step, the DNA isolation, the DNA sequencing. Then that information is run through our bioinformatics pipeline with the algorithms and the neural networks and the AI enhancement, and that all has produced fewer and fewer errors and more and more reliable results.

For instance, we’ve dropped our no-call. We used to get 4 or 5 percent no-call rates. Our no-call rates on first pass are now around 1 percent or 1.5 percent. And if we allow for one redraw if we have particularly low results, the no-call rate becomes less than 1 percent. So, that’s a fantastic situation because as I said, if you draw at 10 or 11 weeks, the fetal component of the maternal circulation is a very small portion of that. It’s amazing that we can get such a high rate of calls with such a small fraction of cell-free DNA. So, then the report is generated after the informatics and that’s returned to the referring physician and shortly thereafter, it’s made available under certain circumstances directly to the patient. And then we, of course, provide counseling and information support whether that be tele- genetic counseling, face-to-face genetic counseling, and in some practices, we have chatbots for both the pre- and -post-test phases of the generation of this information. So, we have a whole panoply of informational support and counseling support for those families and for those physicians to make that information as actionable and appropriate as possible.

Daniel Levine: You’ve recently incorporated AI into the analysis of the test. What’s been the benefit of that. How has that changed outcome?

Paul Billings: Yeah, as I just said, there’ve been really two major improvements. One has been in detection of these kinds of unusual anomalies like the microdeletion syndromes, these are like 22q. The positive predictive value for 22q was historically, with this kind of testing, rather poor. It was in the tens or 20 percent positive predictive value, which is better than nothing, but isn’t as useful as you’d like. With recent improvements with AI that we’ve added to our highly multiplex PCR, many thousands of snip assay, we can get that positive predictive value above 50 percent and that makes it much more valuable. The negative predictive value is also very high, so that’s very important. The other thing, as I mentioned previously, our no-call rate, which can happen because we measure so many different variations in the DNA and so we get a no-cost situation where the sample isn’t right for all those measurements or the amount of cell-free DNA is too low to make all those measurements. Our no-call rate, which used to be around 3, 4 or 5 percent is now less than 1 percent after the second draw and that’s an incredible drop, and that means thousands more of blood samples can be successfully analyzed.

Daniel Levine: This is a test that was initially used to screen women who were considered to be higher risk pregnancies but in 2020, the American College of Obstetricians and Gynecologists updated its clinical practice guidelines to recommend the use of NIPT for all pregnant women, regardless of age. How has that changed the landscape with physicians and payers?

Paul Billings: Well, it’s been a sea change, I mean really universally across the government payers and the third-party private insurance payers. All of them have recognized that NIPT is a far more effective manner for screening pregnancies than previous serum testing was. And that even the American College of Obstetrics and Gynecology recognized that there were differences within the methods of doing that noninvasive prenatal testing. For instance, the Panorama Natera method is much better at detecting sex chromosome anomalies, at detecting triploid and microdeletion syndromes than some of the other methods that can be used in NIPTs for detecting the common trisomies. So, basically all the commercial payers and all the government payers have recognized that average risk women who want to undergo this kind of screening should be offered this kind of testing and that it should be a paid benefit if it’s a third party or government payer situation, and that the children that are identified that are at risk should be confirmed and managed throughout their pregnancies.

Daniel Levine: And is there some kind of regular internal review or an external review about whether a new condition should be added to the panel?

Paul Billings: Well yes, I think both the professional societies through their guideline committees, and obviously the peer reviewed publications through the conduct of large studies and so forth. The innovative biotechnology companies like Natera all look for what additional information can, in a cost-effective manner, be added to the current panel and whether there are effective management measures that make that information highly useful. As an example, we’ve just completed the Smart

study, which was a multi-year, 18,000 patient prospective trial looking at our Panorama method for its ability in the real world to detect anomalies and what we showed, and which will be published in a series of papers in a variety of journals, is that the test is much more effective than the previous serum tests for detecting the common trisomies—that it has a sensitivity and specificity in the 99 percent range, and that this test really does improve in a dramatic way—across different types of cohorts, but in particular, the average risk cohort—the information that can be used to manage those pregnancies.

Daniel Levine: Looking out many years, given where technology is going and the changing cost of things like sequencing, do you ever see a much broader applicability of this test to screening?

Paul Billings: Well, I believe that over time we will be able to do a whole genome analysis of the fetus using a maternal sample and with that information we’ll have a complete genetic picture over time as we learn what the meaning of some of these genetic variations that we would detect really are, but over time, we’ll have a complete genetic picture of a developing fetus at various stages of gestation that could be enormously valuable in management. Obviously how we differentiate significant genetic variation from incidental genetic variation, or not very meaningful genetic variation, will continue to be a challenge and we’ll need better data sets, more diverse data sets, more unbiased data sets to get to that world. But I do think that over time we will have the opportunity to have complete genetic information. And then the challenge will be to make the components of that information useful, continue to offer families and their physicians meaningful interactions, meaningful into ways to manage that information to the benefit of the pregnant mother and to the benefit of the fetus.

Daniel Levine: Paul Billings, chief medical officer for Natera, Paul, thanks so much for your time today.

Paul Billings: Thank you.

This transcript has been edited for clarity and readability.