NEONATAL Genetics!! FISH?! Karyotype? Microarray? Whole Exome Sequencing??

When should you send a fish in the NICU or what does that actually even mean? What about a microarray or whole exome sequencing? Well, we've probably heard and used a lot of these different tests. Today in this video you're going to actually learn what they all test for and when to send each one. Hello, I'm Dr. Tala and I've been a neonatologist for 17 years now and as you all know we deal with a lot of genetic issues in the NICU. Shout out to our members who requested this video. If you want that sort of power then think about joining our membership. Now let's talk about the tests themselves. Before we cover those I just want to say something very briefly and that is just acknowledging the amazing advancements in the last 20 years with the testing of genetic diseases. It really is quite incredible how much faster the testing has gotten, how much more sensitive and also how much cheaper it's become as the process has become a lot more efficient. And like we said, studies have shown that the cost of sending the test is kind of insignificant compared to the benefit of the test. So that's even been shown with the extended genome testing. But, and this is important, even though the technology is available and it's relatively cheap, that doesn't mean that we should stop using our clinical skills. If a baby looks like they have Down syndrome, you don't have to send all the exome sequencing. Or if the baby is floppy and the mother has myotonic dystrophy then just check for myotonic dystrophy. So it's still important that we are discerning in practice. Let's go through the tests. Let's start with one, the karyotype. This is kind of like the basic chromosome testing that we all think about when we're sending chromosomes. So I always tell people it's kind of like that project that we did in high school science where you had to kind of cut out each chromosome and then stick it next to its pair. More of a craft project than a science project, but you know what I mean. As you can probably imagine, this is only going to pick up really big changes in the baby's chromosomes or in the baby's genes. You're not going to pick up a single gene defect on the regular karyotype. But you would notice a whole extra chromosome like in trisomy 21 or you would notice if there are large chunks of the chromosome added or missing to each chromosome. So normally this would have to be in the realm of above 5 to 10 megabases changing in the chromosome to be able to notice it on the karyotype. Cytogenesis also use a type of staining, a Giemse staining, or Giemse, I don't know how it's pronounced, Giemse staining that gives the karyotype each chromosome different bands so that they can more accurately say which part of the chromosome is missing or is added or longer than it should be. And this is called G-banding. You've definitely all seen it. What about picking up deletions with a karyotype? Like we said, it depends on how big that deletion is. We've already discussed that the DeGeorge syndrome is a deletion of about three megabases. So you're not going to pick that up on a karyotype because it's less than the 5 to 10 megabases that we need to be able to spot it. Really, we now use karyotypes to diagnose trisomies. So trisomy 21, 13, 18, or an abnormality in the sex chromosome. So an extra X or a missing X or to see if it's a girl or a boy, X and Y. If you really don't know what's causing the clinical symptoms, then just sending a karyotype is probably not going to be helpful just because it really isn't that sensitive. Maybe the baby could have trisomy 13 and you missed that on exam, but otherwise it's unlikely to be that helpful. But if a baby does have a trisomy, especially trisomy 21, it really is very important to actually get this type of testing. And that isn't necessarily to make the diagnosis, but rather to be able to tell whether this type of trisomy 21 is inherited or not. As in, does the mother have an increased risk of having another baby with trisomy 21? So two different scenarios. A baby has Down syndrome. We get the karyotype and we see on the karyotype that there's a whole extra chromosome. So we have three copies of chromosome 21 and the baby has trisomy 21. This was most likely just a mistake as the egg was dividing and that ended up with two 21 chromosomes in one egg instead of just the one that the egg should have. This happens fairly commonly, especially as women get older. That's why older women are more likely to have a baby with trisomy 21. The other thing that you might see on the karyotype isn't a whole extra chromosome 21, but you could see a little bit of the chromosome 21 stuck onto another chromosome. So the baby does effectively have three copies of trisomy 21, but it's not a whole extra one. What this is much more likely to imply is that the mother had a balanced translocation. So that means the mother had all her genetic material that she needed to have, but maybe a little bit of her chromosome 21 was stuck onto another chromosome. So when her eggs were made, she ended up with too much chromosome 21 attached to another chromosome. So in that situation, if you see a Robertsonian translocation, so you see that the extra bit of chromosome 21 is stuck onto another chromosome, it's important to go back and test the mother just so that she can know, yes, she might be at increased risk of having another baby with a trisomy 21. This wasn't just a one-off accident as the egg was dividing. Generally, when we send chromosomes, it can take a few days up to about 10 days for it to come back. So not too long and relatively cheap. It is kind of like the easiest karyotype testing. Two, fish. Now let's talk about the fish test or fluorescent in-situ hybridization or how we casually say, send a fish. I feel like this confuses a lot of people because it isn't necessarily recognized that you have to know exactly what you are fishing for. Just like when we do actually go fishing, you have to have exactly the right bait for the fish that you want to catch. This isn't just kind of like spreading everything and just seeing what you find. Usually, we send fish testing for the aneuploidies. So to check if there's an extra trisomy 21, 18, 13. We also use it to see if it's a boy or a girl. So we check it for the sex chromosomes, an X and a Y. We can also use it to test for deletions. For example, we can test it and we can use it for DeGeorge syndrome to see if a baby is missing that 22q11 part of its chromosome. So what is the actual fish test? Well, what it actually is, is a DNA probe. So it's a probe that attaches to the DNA that you are fishing for. And if it attaches to the DNA that you are fishing for, then it will light up in a fluorescent way. So for example, if you want to do a fish study for chromosome 18, then there'll be a little piece of DNA that will attach to a specific part of chromosome 18. And if it's a trisomy 18, then you will see three fluorescent lights in your field. This would imply that you have three copies of the chromosome 18. So you have trisomy 18. Again, I want to emphasize this here. A fish is good because you can get it back quickly. But a fish will not tell you if there is a genetic cause for the trisomy because it will just light up. You can't tell if that chromosome is stuck onto another one or if it's a whole extra chromosome. So yes, you can use the fish to get a quick diagnosis, but it won't tell you if there's a translocation and the mother is at increased risk of having another trisomy. We could also use the fish for DeGeorge syndrome. Like we said, in DeGeorge syndrome, there's a deletion of the 22q area. So if you have a probe that just attaches to the q end of chromosome 22, that specific area that's covered by DeGeorge syndrome, and you throw it onto the chromosomal material, and only one light flashes up, it means that you are missing a whole area of that chromosome 22q because obviously you should have two lighting up, one for each chromosome. So you can also use it to rapidly diagnose DeGeorge syndrome or really any other fish probes that have been developed. The good thing about the fish testing is that you are basically taking the DNA probes and just putting it onto the DNA material. So it doesn't have to be in the perfect metaphase where all the DNA is kind of clumped up into the perfect chromosomes and they're all lined up. You can do it immediately on the cell. So it's generally very quick to get back. So normally just like a couple of days. Often with trisomy 21, we can diagnose it just from the physical exam, but often parents really do want that like confirmation with a lab test. So even though sending a fish will not tell us all the information that we want to get from the genetic testing, often we will send a fish as well as a carrier type if it wasn't diagnosed prenatally just so that the parents can have that confirmed diagnosis as soon as possible. Three, single gene testing or genetic panels. Obviously to send these off, there has to be a gene or a deficit that you're more concerned about. For example, in the US, we screen all babies for cystic fibrosis at birth. And if the screening test is then positive, then the state will end up looking for genetic abnormalities in the CF transporter. And by the way, there are about 1,800 known genetic abnormalities that can contribute to an abnormal CF transporter, which is kind of like the channel membrane, which is responsible for sodium and chloride absorption and secretion across membranes. So if you do have a positive screen for cystic fibrosis, then the state lab will test for a few of the most common genetic abnormalities, not all of them. There are obviously so many single genes that we know about that we can test for whether it's cystic fibrosis or different types of surfactant deficiency or arrhythmias, long QT syndrome. So in this case, we have to be very specific and ask for that gene to be tested for. Now what a lot of labs have done is made it so much easier for us by having specific panels for whatever symptom the baby has. So for example, if we have a baby with unexplained cholestasis, so we have an elevated direct hyperbilirubinemia, and we really don't know why, then we can send a testing off for a genetic panel of cholestasis. And that lab will test for every gene that is known to involve a genetic form of cholestasis. So for example, alpha-1 antitrypsin and Niemann-Pick disease and allergial syndrome and all the other ones that have been associated with cholestasis. Another one that we're using increasingly is a seizure panel. So all the abnormal genes that could be involved with like microvascular or microscopic changes in the brain that predispose babies to having seizures. So obviously we send that when we don't have another cause for the seizures. So be aware of these panels and exactly where to send them. Often the companies that actually run these panels are very helpful in making sure that you have the blood sent off correctly and staying in contact with you before you get the results back. And getting the results back, it can be a completely varied amount of time. I would say somewhere between two to six weeks, obviously depending on the panel as well as the lab that you're sending it to. Four, chromosomal microarrays. Up until recently, this was kind of like our high sensitivity test in the NICU when we didn't really know what's going on. But as I'll talk about in a second, this is kind of slowly getting replaced. Microarrays are basically like a high resolution type of karyotype. So they're more sensitive than the regular karyotype in that we're more likely to pick up micro deletions or inversions or small additions or smaller changes in the genome. So it's definitely more sensitive than a karyotype. Chromosomal microarrays use a microarray chip that contains DNA probes. So basically, all of these probes attach to little DNA segments, thousands of them, all over the chromosomes. And these DNA probes will also tell you how many copies there are of the genetic segments as compared to a normal reference. So in a way, it's kind of like a fish because you need a probe to actually bind to that little piece of the DNA. But it's actually just not like a fish because you don't have to say specifically what you're looking for. It just throws on all the probes on all of the chromosomes. In fact, you're testing for something between 1,300 to 3,000 genes. And because the probes may attach multiple times, you can also look for the copy number variance. So areas of the DNA that have been repeated over and over again that may indicate a genetic issue. Copy number variance are not rare. And so sometimes if you get the microarray test back and you're told that you've got however many copy number variance in chromosome eight or whatever, it's difficult to know whether that is actually responsible for the clinical symptoms or not. Ideally, what you want to do is also check the mother and the father's microarray. If they don't have any clinical symptoms, but they have the same copy number variance, then it's unlikely that that is the cause of the symptoms. So often, especially when we have weird abnormalities in the microarray and we don't really know what to make of them, it's important to check the microarray for the parents to kind of see if it's clinically relevant or not. An important thing that you have to know about the microarray, just like the fish, it will just tell you whether you have an extra piece of trisomy 21 or 18 or 13. So a microarray is not the test that you want to send if you are worried about a trisomy 21 or a 13 or an 18 or whatever. It will take a really long time for it to come back and it will not tell you if there is a translocation. It will just tell you if there is a little bit of extra genetic material. So it really won't give you the information that you want. If you want a quick result, get a fish. And if you want to know whether the mother has an increased risk of having future babies with trisomy 21, then get the regular karyotype. Microarrays are definitely taking quicker to come back. It used to be kind of four to six weeks. Now I feel like they take about two to four weeks for them to come back depending on which lab you're sending the microarray to. Five, whole exome sequencing. And this has been shortened to WES. As this has become cheaper and faster and we're gaining more information about it, this is soon probably going to take over the chromosomal microarray as like the test that we do when we don't really know what the baby has genetically. If you watch the first video on genetics that we filmed, then you'll know that the exome is the part of the DNA that actually codes for the protein. So we call it a gene when that piece of DNA is what's needed to code for the protein. Remember that the DNA is in really, really long strands in the chromosome and one continuous strand. And the exomes are like intermittent parts of that long DNA strand. Amazingly, only about one to two percent of the DNA are actual exomes. The rest of it are introns and they don't code for proteins. So in the whole exome sequencing, basically, they are looking at every single nucleotide, every single little piece of DNA that's in every single exome. So really, this is kind of the most sensitive way of being able to identify whether there is anything wrong with any of the genes. It isn't just one probe on one certain area of that DNA, like a microarray. No, it's literally decoding every single nucleotide in that gene in every exome on the DNA. Whole genomic sequencing is where the entire DNA is sequenced, like all of it. So every single base on the baby's DNA is basically being sequenced. So all the 98 percent of the introns as well. Maybe eventually this will have more utility, but where we are right now, where we're not really sure exactly what the introns are responsible for, definitely involved in like upregulating and downregulating of proteins and in repair and other stuff, but not really sure exactly what changes in the introns mean. It's very difficult to rationalize actually getting the whole genomic sequencing unless you are in a test situation. But the whole exome sequencing, where again, it's including every single gene that codes a protein, then really if you don't know what's going on with a baby, then this is a very good place to start or maybe even finish. And now, like I said, the turnaround times are much faster. We can pretty much get some of this back in one to two weeks, again, depending on the lab. Before I end, I just want to say something very briefly about the ethical implications of all of this. Obviously, if you're doing the whole exome sequencing, then you might find other genetic variations that you weren't expecting to find. For example, whether a baby has an increased risk of breast cancer or pancreatic cancer or whatever in the future. Obviously, as a person, I would love to have this information if that is true, because I'd like to be able to do something about it in future. Unfortunately, in many countries, this now becomes a pre-existing condition. So, for example, if you have an increased risk for a breast cancer or some other disease, then maybe it's harder to get insurance in the future. Obviously, we'll have to determine how to deal with all these findings as we move forward. And if the results are going to help us in the NICU and prevent unwanted surgeries and tests and, you know, improve medical care, then we're not going to not send the whole exome sequencing just for some possible future issue with insurance. So definitely from an ethical standpoint, we are going to have to deal with all of this as we move forward. And like a lot of things in medicine, we should be discussing the risks and benefits every time we get a test, especially a genetic test with the family. So this is something that should be decided with the family and letting them know about maybe finding out other stuff that they might not want to know. Okay, so that was our lecture on genetic testing. If you got this far, then please like this video. And if you do want more neonatal education, then please think about joining our channel and becoming a member if you want a say in which videos we put out next. Thank you so much for being here.