Count your chromosomes

Not much time can pass before we steer into the topic of non-invasive prenatal testing (NIPT, but more appropriately NIPS for screening). It is such an exciting field of innovation, and a fairly complex one at that, despite the seemingly simple premise of the test’s purpose: to screen for an abnormal chromosomal count in the baby of a pregnant mother.

But nothing is ever as simple as it may first seem, and NIPT is no exception. Important work has come out highlighting some of the reasons that might lead to false results in an NIPT test, and they are fascinating. In essence, it is the fact that the majority of NIPT tests do not test for altered numbers of all chromosomes, which can mess up the results. The vast majority of NIPT tests look only for problems encountered with chromosomes 21 (resulting in Down syndrome), 18 (Edwards syndrome), and 13 (Patau syndrome), as well as the sex chromosomes. That's for a good reason, as these are the most common unbalanced chromosome counts (termed “aneuploidies”), and adding extra chromosomes into the test reduces the overall power of the test detection as compared to testing for only chromosome 21 levels (the most commonly encountered problem). In fact, when it comes to chromosome 21, NIPT is now considered the most accurate non-invasive test.

Then what is the issue? Well, chromosomal imbalances can occur for every other chromosome in our genome. We have 24 different kinds, 22 that are found in all humans, and two different sex chromosomes, either X or Y. We have two copies of all these, one inherited from mom, one inherited from dad, and women are typically XX for sex chromosomes, while men are XY. That is what is usually observed.

Extra chromosomes can be found, and still can result in a healthy and reproductively viable individual. Extra sex chromosomes are the best example because the biological impact can be very mild. The majority of people with an extra X chromosome will never know it. Another example is Down syndrome, although there is the potential for an entire spectrum of biological outcomes on the individual's health. Indeed, a person born with an extra 21 chromosome has an increased chance of health complications compared to the overall population’s average, but then so do plenty of other genetic outcomes. Everyone will always somehow be affected by the type of mutations that we either inherit or that spontaneously arise, and individuals with extra 21 chromosomes can also have long healthy lives.

So although very rare, aneuploidies with other chromosomes than the ones most frequently tested for, can and do occur, though apparently below the rate of 0.5% of pregnancies. It is the study of these aneuploidies that we want to discuss, as it is the largest published of its kind, and provided many valuable revelations.

Rescuing false-positives

The first revelation was that by looking at the aneuploidies of all chromosomes, you reduce the odds of making a false result during your test, or encountering a test failure. That is because, if other chromosomes are not tested for aneuploidy, but it does exist, then using such data as a normal background can compromise the test. What you think is a normal background upon which to test the aneuploidy of chromosome 21, 18, 13 or the sex chromosomes, is actually not accurate.

How significant is this? While the false-positive rate for trisomies seems to continuously be improved upon, and are now tiny compared to traditional methods, test failure due to the failure of the NIPT assay itself can still be encountered around 2% of the time. Therefore, at least a portion of the existing false-positives could be expected to be rescued. Alternatively, individuals with failed test results using the current NIPT test that do not test for all chromosome anueploidies, can be considered as being at increased risk for trisomies.

Second of all, the discovery of an aneuploidy in any of these other chromosomes appears to be associated with important diagnostic potential, and is most frequently a cause of miscarriage (about 42% of the studied cases), but also factors into other pregnancy complications, such as intrauterine growth restriction and in utero fetal demise. The vast majority of miscarriages were associated with an extra chromosome 15. This knowledge of additional chromosomes could therefore point to possible clinical outcomes. Incredibly though, a number of these additional aneuploid cases still resulted in a normal live birth, most frequently observed with the trisomy of chromosome 7.

Thirdly, these rare aneuploidies accounted for less than 77% of the abnormal NIPT results. Extra chromosomes are not the only chromosomal anomalies that can contribute to proper measurement problems during the NIPT test, as the genome can be very complex in its function, and sometimes can be rearranged in a wild fashion. For example, copy number variations, a jargon used to describe the fact that certain fragments of our genome can exist in multiple copies, affected the results in more than 10% of the cases. Some diseases are developed from these extra copies of genome segments, because too many copies of a certain gene product will be produced accidentally, putting out of balance the existing harmony of normal cellular functions.

In more than 13% of the tests, multiple types of problems were found within a single sample, such as multiple forms of both CNVs and aneuploidies or other genomic imbalances (the same chromosomes having vastly different structures). Such chromosomal imbalances, termed “dysploidy”, could be indicative of maternal cancer, so that could prove to be another finding of the NIPT test. In this study, the dysploidy accounted for about 4% of the abnormal results but it was not known if these mothers actually did have cancer or not.

The point is that the production of a healthy baby is not a trivial process, and many obstacles can exist along the way in the form of an affected genome. Age appears to be one influencing factor, as these abnormal results were most frequently observed in older mothers. And aging mothers are not the only ones to blame. It has been well-known for a long time that older men have an increased likelihood of having children with developmental disorders, and a new study recently quantified that risk as being roughly 4 times more likely to pass on mutations to offspring by aged men than women. So it doesn’t pay to keep those gamates unused for too long in your life, or you run the risk of passing on to your child 0.37 of new mutations per each additional year of maternal age, or 1.51 per year if you are a procrastinating father-to-be.

Take a breather.

Because it gets even trickier.

Fetal DNA or not fetal DNA?

For a number of these pregnancies, amniocentesis results were also available, which is the invasive procedure considered to be the true gold standard diagnostic procedure. In the amniocentesis, a sample of the amniotic fluid is collected as it is considered the true fetal representation of the genome. The NIPT procedure doesn't actually test fetal DNA directly, but rather that of the placental DNA. It is the placenta cells that shed the DNA into maternal blood, so the fetal blood is not exposed to maternal blood directly. Typically these genomes are supposed to be the same, as both the fetus and the placenta develop from the same progenitor cells, but this might not always be the case. We previously discussed this topic in the introductory post on NIPT. Depending on the timing during the early development of the fetus, mutations can occur that could be confined only to the placenta and not affect the fetus. In such cases, it is referred to as “placental mosaicism”. NIPT could be detecting such mutations in the placenta whereas the fetus could be just fine.

Such discordance was observed in this study 13% of the time, where the amniocentesis results were normal but the NIPT results were indicating a variety of different chromosomal trisomies. This is the reason why it is important to confirm a positive NIPT result because the baby could be fine. Nevertheless, the finding of such a mosaicism result with NIPT is still important because placental mosaicism can be associated with pregnancy complications, and hence might require special attention. In most of the cases, the baby was fine when born, but cases of interuterine growth restrictions were also observed.

Continuing on the topic of mosaicism, in rare circumstances maternal autosomal mosaicism was detected using NIPT in otherwise unsuspecting and apparently normal mothers. That is because the non-invasive prenatal test was detecting trisomy at a fraction much higher than the average total DNA count that could be attributed to the fetus alone, meaning, the mother's DNA was the source of trisomy. And in one case it was chromosome 8! Once again, individuals with extra chromosomes that were normal and apparently healthy.

What women want

It was also interesting to see the reasons why NIPT tests were provided to pregnant women: for more than 80% it was a primary screening method, 15% were assessed due to advanced maternal age, for 4% NIPT acted as a follow-up of combined first-trimester screening results, and for only 1% of women the screening was offered due to previously observed fetal sonographic abnormalities. Thus, for the vast majority of women in one of the studied cohorts, NIPT was offered as the primary screening method of choice. With such a high number of women electing for this type of procedure, proper education and understanding of the test benefits and limitations should be paramount, including the type of test that is most likely to provide most informative results. As the authors of the paper noted, "we recommend that patients should be given the option of receiving test results from all chromosomes."

Adapted from Pertile MD et al. 2017. Sci Transl Med 9(405). pii: eaan1240

And the complexities of prenatal DNA screening do not stop there, including what type of information could be probed for, which can exceed that of aneuploidies and include testing for specific genetic diseases. These are not as readily available as NIPT, which is one of the most widely adopted molecular tests in history, but the boundaries of detection are being pushed all the time. If you are interested learning more about these possibilities, please contact us at Merogenomics.

The bottom line is that NIPT tests are not as simple as the public - and even the providers of these tests -might think, and it appears that to help mitigate some of the potential problems that could be encountered with the test provision, ideally you should be looking for those tests on the market that offer testing for aneuploidies of all chromosomes. This is important, considering that the follow-up tests to a positive NIPT result are the invasive tests that women aim to avoid in the first place. And judging from the trends regarding how often women now use NIPT as a primary screen, these are some important considerations.

This article has been produced by Merogenomics Inc. and edited by Kerri Bryant. Reproduction and reuse of any portion of this content requires Merogenomics Inc. permission and source acknowledgment. It is your responsibility to obtain additional permissions from the third party owners that might be cited by Merogenomics Inc. Merogenomics Inc. disclaims any responsibility for any use you make of content owned by third parties without their permission.

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