Pregnancies just got new test to study fetus
The world turns around, seasons come and go, storks migrate back and forth, and women get pregnant. Nothing has changed in that department for countless millennia. What has changed, quite recently, is how pregnant women have started applying genomic technologies to assess the health risks of their babies, even prior to being born. Colloquially known as NIPT test, which stands for a non-invasive prenatal test, it is a term that is rapidly becoming familiar to pregnant women.
How rapidly you may ask? According to the data by Dr. Xin Jin of the BGI genetic company which was presented at an ASHG2016 conference on human genome sequencing, NIPT is the fastest adopted molecular test in history. The current estimate number of women that have taken such prenatal genetic testing is around 5 million in the entire world. That is a pretty solid number. What is amazing though is that more than half of these tests, around 3 million, have been done in China alone.
So China is definitely leading in this area! Well, combine the most populous country in the world with a policy of one child only per family, along with the seemingly ever expanding economic status of the Chinese population, and perhaps this result should not be so surprising. What is amazing though is that half of these tests in China were performed by a single company, the renowned BGI, which by now has a global status and presence. If you haven't heard of them, it's only because genomics testing is still not your dinner table topic du jour, but if you are immersed in genomics, then you would definitely know about them.
Why is NIPT taking world by storm? Because it is touted as a non-invasive test replacement for established invasive procedures such as the collection of amniotic fluid or chorionic villi sample. These tests are used to determine if chromosomal imbalances are present in the fetus, such as Down syndrome, the most common and most feared of all developmental pregnancy abnormalities. Which by the way, such non-invasive pregnancy chromosome test is not a replacement, just a cheaper and safer alternative, though not exactly matching the accuracy of the former tests. And it doesn't test your fetus DNA directly, but that of the placenta. As they both originate from same source, the fertilized egg of course, they are looked at synonymously, but there can be rare and unusual complications associated with such DNA source. But from a pregnant woman's perspective, I can see why it is greatly appealing: take the test, which only requires a maternal blood draw, and figure out if all chromosomes are looking normal or not. If something looks fishy, then you can go for the invasive test to confirm. So in simple terms, it is a blood test for birth defects based on chromosome testing during pregnancy.
How the fetal DNA is analyzed for chromosomal abnormalities can potentially vary, as different technologies could yield different results. But what all of these tests have in common for sure, is the source of DNA. The DNA is released from broken up placental cells. As placenta is awash with maternal blood, these DNA fragments released from cells can freely circulate in blood. Hence the test can also be termed cell-free NIPT.
But now a new alternative has been established which poses greater advantages to current cell-free NIPT tests. This information was also presented at the above mentioned conference by Dr. Amy Breman of Baylor College, and the moment I found out about it, I ran to her to learn more details! Well, it's not exactly some latest secret; all the information on this new prenatal pregnancy test has been published already. But it could be transforming our current approach, also it is pretty cool and high tech so I have to tell you about it. Yet another reason to look forward to those storks appearing in the sky!
So what is the primary difference? These researchers, instead of going after the DNA fragments that are circulating in the blood, looked for entire intact cells. Just as cells can release their contents into the blood stream, whole cells can be dislodged and go for a bodily ride in the highways of our circulatory system. For this reason, the authors referred to it as cell-based NIPT, and it is the world’s first successful demonstration (and not like it hasn't been tried before - these guys just got it right!) In our interaction, Dr. Bremen remarked that “researchers have been trying to make cell-based NIPT a reality for over 30 years, and we are thrilled that we have reached a point where a clinical test might be available in the near future. We are driven in part by the knowledge that there are many genetic disorders more severe than Down syndrome, and in order for a non-invasive test to become a new gold standard for expecting mothers, it should be able to detect everything that an amniocentesis or [chorionic villus sampling] can detect. By using intact fetal cells, we think this new test will be able to accomplish that.” Imagine that! Being the team that cracks such a long time challenge.
So the procedure still only requires a maternal blood draw, from any innocuous body part of your choice (well, obviously usually just your arm, but you could always insist on something else), and a series of complex procedures are undertaken to identify the correct fetal cells out of the organic blood mixture. It was the use of antibodies to identify cells by their specific molecules found on the outside of the cells. In this case it was cytokeratin, which acts like a key and lock mechanism. The cytokeratin on the cell surface being the key inserting itself into the keyhole presented by the antibodies, allowing you to fish out the type of cells you are seeking. Fancy tools like a needle with a 40 μm ceramic tip are used to pick up these cells. To give you some perspective, your average hair would be around 60 μm (and of course that is just an average, some having hair of type that resembles an evolutionary missing link, and some so fine that you think they shave their entire body for some strange reason).
Tricky stuff, and in the end the average yields are 0.7 cells/mL of blood. It is almost like looking for a needle in a haystack. Liquidy biohazardous haystack that is.
It doesn't seem like much, and it isn't, but you have to understand how powerful these technologies are. A single cell now provides enough genetic material to perform serious analysis, as has been demonstrated in the recent past, including cancerous cells. So the advantage here is that instead of fishing out broken up fetal material from amongst maternal contamination, you can isolate a whole intact fetal genome from such cells, so I hope you can see the difference. It's like buying a car versus car parts mixed up with a plane parts, and you don't know if you even have all the parts until you put it all together and stick a key into ignition to see if it works or not.
Such whole fetal genome was then amplified, which is a fancy way of saying making multiple copies of, and then assessed for its content either using microarrays or genome sequencing. Arrays use complementary tags to find a specific section of genome. Think of it as molecular velcro, and it can be made to assess contents of anything in the genome, in any or all of your chromosomes. So if something is duplicated, or is missing, those tags will show that a signal is increased or decreased. So now not only can you look at if all chromosomes are there in correct number (so no trisomies of any kind), but even if pieces of chromosomes might be missing or duplicated, which can also lead to developmental complications and abnormalities.
With genome sequencing, you sequence the whole thing, align it to a known human reference so you know what is what and where, and then you start comparing how many times a given section of a genome was decoded in the process. Imagine if you took fragments of the same puzzle image from many boxes, had it all mixed up, and then using an image of one of these boxes, you put all of them back together. Because you have puzzles from many boxes, some of the images would be pieced together more than once. It is like that with sequencing where a single puzzle piece is a single small fragment of DNA. Again, if something is duplicated or missing, it will be observed with a higher or lower frequency than the average for the rest of a chromosome. So the power of cell-based NIPT is much more refined than the typical cell-free DNA NIPT. The authors showed that they were able to detect missing sections of just under 3 Mbs of size, which is much smaller than current detection limits of cell-free DNA NIPT. For reference, your entire genome is 6 Gb in size, where Gb refers to a number of nucleotide bases in your genome, at 6 billion bases. So tiny fragment changes can be investigated, and this resolution is likely to only get better. Such small alterations are currently just not detectable reliably enough using cell-free DNA NIPT. It can be done to a degree, but the accuracy is just not up to snuff.
And the more fetal cells that can be isolated, the more individual assessments can be done, which not only validates the results, but also allows for the discovery of some funky results that under a regular cell-free DNA NIPT you would get an incorrect answer. As I mentioned, the source of circulating DNA in mom’s blood is not directly from the fetus but from the placenta even though I use these terms interchangeably (as does everyone else). Same with those whole intact cells. This can be problematic if the placenta is not homogenous, meaning it is made up of cells with only one and the same type of genome. Sometimes in this super-early development a mistake can occur in the subset of cells which will remain in the placenta without being present in the fetus. This is called placental mosaicism and occurs roughly in 1-2% of pregnancies. In about 10% of such cases the fetus cells could be affected as well. Yes, technically you can be born with two different genetic materials! I recounted one such scenario in a previous post.
Adapted from Ledbetter DH et al. 1992. Prenat Diagn. 12(5):317; Phillips OP et al. 1996. Am J Obstet Gynecol. 174(3):850
So now imagine a scenario where the placenta DNA tells you one thing, but the fetal information is actually something else, then you are obviously going to get a wrong result. If you are just taking a mixture of these DNAs directly from the maternal blood, you can't tell if it is a result of mosaicism or a genuine fetal problem. This is one of the reasons why you always always always have to confirm a positive NIPT test result! While chances are small, it simply could be wrong information, so you can’t rely on it. Not to mention that all such serious information of the fetus being affected should always be validated.
But if you have intact cells available, you are no longer looking at a big mixture of random DNA fragments, but rather can assess those cell genomes individually. If enough cells are isolated from the blood and looked at, then if mosaicism is present, you could see it by observing two different types of genomes isolated from different placental cells. And it's not like I'm going all theoretical on you here; these authors actually found and demonstrated such a case! The use of intact cells allows one to potentially identify cases of placental mosaicism, or to discover if you are the rare individual who might be composed of more than one type of genome!
And there are even more benefits! Having access to the whole fetal genome as opposed to a mixture means that the actual sequence could be analyzed with greater accuracy than would be possible with circulating cell-free DNA. As you could imagine, the majority of circulating DNA in maternal blood will be of maternal origin, and although the fetal amounts can vary depending on many factors (gestation age, weight of the pregnant woman), typically the fetal amount is around >10%. So by the time you are done sequencing this mixture, and figuring out which fragments are fetal origin and which ones are maternal, your average read depth of the fetal genome is 0.1X. Do you know what the minimal standard required for clinical grade is? 30X!
According to Dr. Xin Jin of BGI, 93% of these fetal fragments get to be sequenced only once! It's okay if you just want to determine if there are chromosomal imbalances because many such fragments would make up a content of a chromosome, but if you want to start getting specific sequence data, we would be talking about some serious uncertainties. If a mother has a certain novel mutation that she wasn't born with, you would not be able to tell if it's her or the fetus! Or, if a mother has cancer, this can also offset the results, suggesting a chromosomal imbalance if not properly identified. And that's another consideration, the current NIPT tests could be identifying cancer development when a parent least expects it. Maybe that's a good thing, maybe not, depending on whether your body would be able to heal the problem or not. Obviously I would consider such knowledge better to possess than not, but that is my own personal opinion.
Dot dot dot, now we are going back to intact cells again and the benefit of access to a pure fetal genome. Such genomes allow for more precise sequencing and an ability to distinguish those mutations that are not present in maternal DNA, and vice versa. As a good example for the use of such a procedure, the authors mentioned that novel mutations that naturally occur with any pregnancy can also affect the ~500 genes currently known to cause severe intellectual disability, a phenomena happening at a frequency of 3–5 times higher than the incidence of Down syndrome that NIPT is primarily advertised for. I hope you can see why I got pretty excited about this information, and I truly hope this technique takes off even faster than a dislodged placental cell in the stream of heart propelled circulating blood!
But you can still get some amazingly powerful information from NIPT. Going back to the presentation of Dr. Xin Jin at the ASHG conference, that while on individual basis NIPT is not powerful enough, on aggregate bases, Chinese researchers have shown some amazing data! They were able to identify population-specific rare variants, even at such low frequencies as mutations observed only at ~0.2% of the time in the population! Such data is always useful in understanding how different populations are distinct from each other, and therefore what unique considerations should be taken into account when analyzing their health data. For example, they were able to show that some of the existing database knowledge we rely on for the interpretation of disease development or drug response, is not accurate with regards to specific Chinese populations. This is how a continuous build-up of knowledge helps refining the state of knowledge that future generations will be able to rely on.
But enough side tracking! Going back to gloating about cell-based NIPT, it’s not as though you would be free of any of the issues mentioned above for current NIPT approaches. Here is one that is probably going to take you by surprise: did you know that you can have cells of fetal origin circulating in the maternal blood for years after the delivery? One example that the authors of the study cited indicated that male cells could be isolated from a woman's blood even 27 years after pregnancy! How about that?! When moms say they are connected with you for life, they really are not kidding! I guess you never can deny your biological heritage. But the authors also pointed out that such cells would not be isolated using the antibody capture methods employed in the study, plus such cells from former pregnancies are really rare in the first place, so they should not be a confounding factor in the currently discussed methodologies.
In the end, the authors probably summarized this novel technology the best: "the results suggest the possibility of developing a cell-based form of NIPT with ability to detect abnormalities with a similar accuracy as can currently be obtained with amniocentesis and chorionic villi sampling." I certainly wish them luck as it seems to bring many benefits for those pregnant women who will want to be tested, and as long as the world keeps on turning, and as long as seasons keep changing, and storks keep on flying, let us hope that only women will keep producing our babies.
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