Let's go on then. So basically I'm doing neurogenetic focusing on four different diseases that we do do testing in the molecular laboratory. I'm going to start out with muscular dystrophy and I do have some clinical background on here but we'll go through these quickly to get to the technical how do we test, how do we interpret. So muscular dystrophy and this is with a DMD gene. There's a whole bunch of different types of dystrophies but for this gene DMD Duchenne is the more severe form. Becker is a milder. It is X-linked recessive about 1 in 3,500 males and signs of muscle weakness by about age 3. By early teens really hard respiratory muscles are affected and the survival rate still isn't high but I think there's actually some good therapies and trials to at least make DMD more like a Becker and a milder course of action. One of the points of this is that many of these, a third of these, are de novo cases. The creatine phosphokinase levels are actually one of the other diagnostic tests for this or at least to check the levels and you can see that in males the serum CK concentrations are going to be very high. In females it's not going to be quite as high and sometimes a bit more difficult to identify female carriers from it. The dystrophic gene, it's a very large gene, 79 exons, 14 different known transcripts, 8 independent tissue specific promoters, differentially spliced, so a lot of things going on with this gene. Basically at this point it's like I don't want to singer this, it's too big to singer, we just go straight to next-gen even for just this gene. Here it is, here's showing some of the different tissue specific transcripts and the splicing. So you need to be aware of that but most of the time we go with the major transcript with it. The mutation spectrum and I call this mutation spectrum because the pathogenic variant spectrum doesn't sound quite so, doesn't roll off the tongue as easily, but you can see there's a lot in a lot of different places. But gross deletions over a thousand. This is from the HGMD biobase. Muscular dystrophy, Duchenne, is 2,000. The variants that have been associated with Duchenne and Becker is over 800, so you can see it's not an absolute genotype-phenotype correlation either. And then the ones that have been associated with Becker, most of the time the Becker may be an in-frame type deletion or a duplication which is going to disrupt, have less disruption on the protein. But nearly 4,000 variants detected and all kinds. So gross deletions, gross insertions, duplications, complex rearrangements. It's not a repeat disease. But one of the points is given this mutation spectrum, what's our first-tier test? Can you see them enough? What would you do first? Somebody comes in to test for muscular dystrophy, DMD. Okay, we're going to do deletion-duplication testing first, not sequencing, because we're going to have a greater chance of identifying a deletion or a duplication in the gene. But then if that is negative, then we go on for sequencing. So deletion-duplication. A deletion of one or more will pick up about 64% of Duchenne, about 85% of Becker. Duplications are a little trickier because you can't always assume they're a tandem duplication. So if it is a tandem duplication of an exon, then it may disrupt the reading frame, but you can't assume that. That duplication could actually have happened pretty much anywhere in the genome. Now often things have been shown that they are tandem, but I just wanted you to keep that in mind. It says mutation scanning, but I think we basically go to sequence analysis. Looking at the rest of it, so we'll be picking up, you know, 25 to 30% more for Duchenne and another 5 to 10% of Becker. And the computer is thinking. Now, it says that it's not responding. Now, it responded. So one of the ways, and one of the early ways that they started looking at muscular dystrophy is to doing a multiplex PCR, looking at each exon, amplifying within or over each exon. So you have a whole bunch of these. Sometimes you drop out a small exon and then run them on a gel. And then you'd look and say, OK, these are missing here. These are missing in this person. Now, they're not always designed to be exon 1, 2, 3, 4, 5, 6, et cetera. So these are not always going to be in numerical order per exon. But more than likely, it'd be like if you need to do two gels, the first part of the gene on one, the second part of the gene on the other. What you could see is that these are going to be a little bit more difficult to identify carriers. Because carriers, you're going to need to start looking at intensity of bands. Not easy to do. You can do that. But you'd need to be looking within. So if you say, OK, here's these two here. This one looks a little bit fainter. But what about normally? Is normally that exon a little fainter? So you can do some algorithms. You can have instruments that can detect fluorescent levels. And so you can do this. But carrier testing is a little bit more difficult to do by this gel-based. There's other methods, such as here's a muscular dystrophy on a male from a targeted CGH array. And you can see here, there's several. So here's a deletion of exon 79. Here's a deletion of exons 17 to 44, et cetera. So this becomes a bit easier to see. Another way to do this is multiplex ligation probe amplification. Do you need me to go over what MLPA is and the? OK. What was that? Can somebody help me with the drawing, maybe? Because I just realized that I had kind of assumed that this would be covered elsewhere. I don't know why I assumed that, because I realized I didn't put it in. So multiplex means you can do a whole bunch at the same time. Ligation means you're going to ligate or combine two probes together. Probe amplification. So the point is that it has universal primers. Universal primers, so it'll amplify all of the different ligated probes at the same time. The reason this is important is that you don't need to worry as much about PCR efficiency as you would if each exon had a different PCR primer, then you would need to worry about efficiencies. We're trying to do quantification here. So all of a sudden, efficiencies become very important. So we have our probe that is going to be sequence specific. Let's say this is our exon, and it's going to be placed within the exon. We're going to have a stepper sequence to make these different sizes. We're going to have our universal primer sequence that then we can amplify all with one primer set. So now the fact that we're going to have different sizes is also going to affect PCR efficiency. The bigger the fragment size, the product size, the less efficient that's going to be. So you're going to see a general downtrend of the PCR products with the larger fragments, but you can take that into account with normalization, et cetera. As the probes hybridize, if there's something to hybridize to, you then do a ligase, which will ligate these two together. Once these two ligate together, the PCR is going to be able to amplify over that fragment. If these are not ligated together, you won't get a PCR product, right? So only when there is a DNA sequence there will you get amplification. If you don't have that sequence, and this is all blank, then instead of a fluorescent level like this that you're detecting, you would either get a fluorescent level about halfway, about half for one copy, or basically zero. And for the same muscular dystrophy, if it is a male. Why didn't somebody tell me my paper wasn't in the screen? For a male, you're looking for zero copies, because he only has one X chromosome. You're looking for zero. For carriers, we're looking at the one instead of the two copy. Does that help? OK, then you run this on a capillary, and this is where you get all of these peaks. Is there an internal control that makes sure that? So we know about the PCR efficiencies with the larger products. That is taken into account and normalized for. So that's taken care of. What isn't taken care of, and what you really need to be aware of, is if you have a single probe showing a deletion. So for example, this is what it would look like here. We have to do this in two sets of reactions because it's such a big gene. We have a whole bunch of control genes on here as well to do comparisons. And all of these are falling within the two copy numbers. This is for a male. These don't, but there's three. Now, as long as these three are all within numerical regions, so this is exon 5, 6, and 7. Now we have three probes, so we're going to be pretty confident that this is a true deletion. If we only had one probe there, it could be a single exon deletion, but you don't know. So you need to sequence to see if there is a variant. Because if there's a variant right at the ligation site or close to it, it's going to prevent the ligation. It's going to prevent the amplification. So one possibility of a false positive single exon deletion is that there's truly a variant instead. Now, that variant may be pathogenic or it may be benign. You have to sequence it to find out. Let's think about this. Let's say I have one variant or one probe, one exon that's dropped out on MLPA. I try sequencing it for ML for Duchenne muscular dystrophy. What's the challenge I'm going to face? There we go. If it's truly deleted, it's just going to fail PCR and fail sequencing. And so in this case, it's like, oh, well, our confirmation is that we can't sequence it. Now, if you can sequence it, and you can see a variant there, then you have your answer. But many times, it's like you really have to take the failure of sequencing as your confirmation. A little disconcerting, but that's the way we have to do it. Any other questions? Multiple exons are done in one test, okay, in fact that's how you buy these kits, that's how they're set up, yeah. And I cannot tell you how many times we find a variant that does interfere. I'm sorry. The question was, is it standard to confirm a single exon deletion by sequencing or some other method, I will say, but yes, that is standard. Okay, next question. Would the failure to sequence be equivalent to a deletion on a targeted array? Yes, I think so. I haven't thought that through, but my initial impression is yes. So a targeted array, I would hope though that the targeted array would have more probes. If it still goes back to one probe, then no. But if the targeted array has 10 probes you're looking at and you can see with more than one probe, I would then say that that is a possibility. Okay. I'm going on, right? Okay, it is expected to predict whether it's an in-frame or out-of-frame. So using the previous MLPA results, the deletion of exons 5 to 7, and this was from this Variant Explorer, which I really love. Would you predict this to be an in-frame or out-of-frame variant? And let me tell you, each one of these kind of tells you what that last base is. So there's this, this, or straight. And so it needs to fit into the next exon. You can do this numerically and type them in and you'll get a nice little result. This is just so visual and I never know if these other places want it to be inclusive or not inclusive. Do I put in 5 to 7 or do I put in 4 to 8? And because of that, I like the visual. Okay, so doing this, is this going to be an in-frame, predicted to be in-frame or out-of-frame? Okay, so it's predicted to be out-of-frame. Now, one of the things is that these probes are not always in the exon. Sometimes the exon are too small to fit these probes in or don't have the sequences to be able to do this with. So sometimes the probes are a little outside of the exon. So just be aware that sometimes you're not testing the exon. It may be close to it and practically speaking, it may be. But in those cases and depending on how far away it is from the exon and if the probe goes into the exon or not, you may want to be careful about doing these predictions. Okay, so the HGVS nomenclature can be something like this. C.265 to a question mark, meaning we didn't identify breakpoints. We know this is the exon before. And here is an exon. It's in between these regions. But we don't have an exact breakpoint. We may know that it's in an actin binding domain or a specific domain. We know a frame shift and from that frame shift, from the exons we are going to predict potentially a protein. I don't usually try to predict proteins from deletions. I think it's clear enough that we call it exon deletions. And then give whether it's a frame shift or out of frame. Physicians have usually been very happy with that. But to get this into an HGVS nomenclature, we may need to go further than that. Okay, so if we don't get a deletion or duplication, a causative duplication on deletion duplication testing, we go into sequencing. And as I said, I'm doing mainly massively parallel sequencing, NGS type sequencing now. Although it's still possible with Sanger. It's just, you know, you're going to have about 80 different amplicons. This is actually taken from ClinVar about the variation types. There's 147 conflicting interpretations in it. But benign, likely benign, etc. The molecular consequences, many of them are frame shift. But missense, those are going to be the most difficult to interpret if they've not been reported before. And then some variant lengths. Anyway, just some interesting information for you. Do we need to do a Bayes analysis for Duchenne muscular dystrophy? We don't typically in the laboratory. If you need something to practice on, this is maybe a good one. And I've just included it here. What is the risk based on the pedigree structure? Okay, this person is a known carrier because of two affected individuals. That means it's not de novo or very likely not to be de novo for a de novo thing to happen twice. So she's predicted a carrier. Her daughter wants to know what is her risk of being a carrier. What is her risk of being a carrier given this? It's 1 in 2, right? So her probability that she is a carrier is 1 in 2 and her probability that she is not a carrier is 1 in 2. Okay, the conditional probability given that she has 1, 2, 3, 4 sons all unaffected. So each son has a 50-50 chance of getting the affected chromosome, right? So it's 1 half times 1 half times 1 half times 1 half. So this is the risk to have 4 unaffected males is 1 in 16. So the conditional probability of being a carrier is 1 in 16. The conditional, if she's not a carrier, what's the probability that she'll have 4 healthy sons? It's 100%. So the joint is multiplying the prior and the conditional. Prior and conditional. And the posterior is the joint divided by the sum of the two joints. The joint that she's a carrier, 1 in 32 divided by 1 in 32 plus 1 in 2. So 1 in 2 is 16 out of 32. So it is 1 in 32 over 1 in 32 plus 16 out of 32. Cross out all of the 32s and you get 1 in 17 probability that 2, 2 is a carrier and 16, 17 that is not a carrier. Be careful what the question asks. If the question asks what is the risk of this person being a carrier, you need to add on 1 in 17 times 1 in 2. Okay, let's go on. Anything with muscular dystrophy? Comments? Let's go on to SMA. Onset predominantly in infancy. Limb and trunk weakness, muscular atrophy. It's autosomal recessive incidence about 1 in 10,000. Carrier frequency is about 1 in 100. This now that CF is no longer considered a lethal disease. This has been considered one of the more common monogenic causes of death in infancy. What I can say now is that there is a treatment for it and so that number is going to go down and this will no longer be true. Spinal muscular atrophy, so there's a severe form. Age of onset within the first six months. A neurologist can tell you that they can start identifying signs within a month of birth. Okay, this is something you want to get on quickly which is why there's now newborn screening for SMA, which is an exciting thing. Type 2 is less severe and type 3 is less severe still. And so there are individuals with type 3 that, you know, we are testing that are in their 30s and 40s. This is a complicated locus. There are two genes. One is SMN1, which is also known as the telomeric one for your cytogenesis. You may like that. I like SMN1. And then SMN2 or centromeric one. They have a very high homology. And the differences are only a couple of different variants between the two genes. But these differences are pretty significant. So one is an exon 7. There's another one in exon 8. This one in exon 7 actually is a splicing variant. It affects splicing. So SMN2 splices out exon 7 the majority of the time, not always. The splicing is just, it's not completely efficient with it. And so sometimes you'll have that exon 7 in. So sometimes you'll have something functional. But most of the time, this just is not going to work as well as SMN1. But this becomes very interesting that SMN1, most of individuals with SMN1 are because they have a homozygous deletion of SMN1. Now SMN2, the number of copies they have relates to the severity. So we call SMN2 a modifier of the disease. So if you have two copies of SMN2 and zero copies of SMN1, you're going to have severe form of the disease. But if you have two copies of SMN2 and, sorry, zero copies of SMN1 and three copies of SMN2, then it's going to be less severe. And if you have four copies of SMN2, it's going to be less severe still. Kind of the most I've seen, I think, is eight copies of SMN2 in one individual. Again, the phenotype isn't 100% correlated, but in general. If you look at this, this looks like, well, it's kind of in the same position as of SMN1. Sometimes these are gene conversions, okay? And so maybe this started out as SMN1, but the critical portions of the gene have become SMN2. And so we call it SMN2. Mutations in SMN2 or a homozygous deletion of SMN2 has no clinical consequences by itself as far as I know. Here is a restriction digest for it, and this was the first test. And they're looking at exon 7 for that splice variant as well as exon 8. And here's exon 7 and here is exon 8. And you can see the normal bands will have a 180 and a 156. SMA patients will have 156 only. So here's affected. These are carrier. And here's affected. Here's a normal. And then exon 8 is basically using it to confirm. I'm going to throw this out. I did not see it when I was actually teaching another course. Somebody in the audience came up and said, this happened. He tested this individual with a progressive neuromuscular disease. And he did this by MLPA and found by deletion testing that there was only one copy of exon 7. Okay. So what would be your thoughts of having one copy of exon 7? So most likely carrier only, not affected, although there is a chance that one copy has a sequence variant in it that is pathogenic. And so the recommendation would be if this patient has symptoms to go into sequencing. But then they did an exome test to see if they could identify something. And the report came back as a homozygous deletion of exon 8. Now in this case, they tested only exon 7. What are the explanations? So if you do it by exon by exon, it may be one copy of SMN 1 and two copies of SMN 2. If you look at this, exon 8 may have zero copies of SMN 1 and maybe it has three copies of SMN 2. I wasn't told that. What's the concern there? A gene conversion at or before exon 7 turns SMN 1 into SMN 2. A gene conversion event that affects only exon 8, is that causative? I've actually seen a number of cases by MLPA. If we look at both exon 7 and exon 8, that on exon 7, it's one copy and two copies of SMN 2. And then exon 8, it is zero copies of SMN 1 and then three copies of SMN 2. I've seen that result before. I haven't called it affected because the definition of SMA is a homozygous deletion of exon 7. So what do we do about this? They didn't find any other sequence variants in it. But he does have progressive neuromuscular disease. But I see this all the time in carriers, unaffected individuals, a zero copy of exon 8. Anybody going to give me an answer? Jerry, are you going to give me an answer? What? Yeah. I don't know the answer. I did talk to Tom Pryor, who is kind of the world's expert on this. Not about this case. I was kind of hoping he would be here and I could discuss this with him. But he had said, well, that's true that, you know, it's really the deletion of exon 7. That you need to be looking for. Interesting. Now that there is a treatment, Spinraza, I believe is the brand name for it. And it basically uses an oligo type mechanism to allow proper splicing of SMN 2. So exon 7 gets put back in into at least a number of the cases. And so then turns SMN 2 to function more like SMN 1. And it would at least reduce the severity. I will tell you, this is the one of the most exciting advancements in genetics that I've ever seen. Because it was, I really didn't think that we would see these types of therapies in my career lifetime, and I'm so excited that we have. This was shown at ACMG a couple of years ago, of the clinical trials, and an SMN, an SMA baby that was identified that would have been predicted to be a very severe form of SMA, was started very early in life, and at age two, I guess three-ish, was riding a tricycle. And I will tell you, there were a lot of teary eyes in that room of MDs and PhDs and genetic counselors. It was pretty impressive. So this is the difficulty. We still get difficult cases like this. What do you do? Reach out to others. Reach out, you know, get to know people. Reach out. Discuss this. Have people you can call. Pick up the phone and call the expert in the field. Find the name on a paper and email them. You know, none of us mind getting questions and asking, what more can you tell me about this? We also do carrier testing now for SMA, looking for one copy. I do need to tell you this. To get the drug, you have to have zero copies of SMN1, and some drug companies will say at least one copy of SMN2. Well, that's true, because if you don't have any copies of SMN2, the drug's not going to work, right? And so basically, it is now that if an individual is picked up on newborn screening to be homozygous deletion of SMN1, then you need to do and get a copy number of SMN2 as part of their diagnostic workup. So the same thing. We're using MLPA in this case. And here we're looking at exon 7, sometimes exon 8. There's two different kits that can be used, and then an SMN2 exon 7. So we're looking for carriers, so one copy. Now, sometimes you see this. We call it a potential silent carrier. In some populations, there's a known variant, two variants, that are known to be on the same chromosome, same allele, as two copies of SMN1. So when we, as a molecular test, only test for copy number, we say we pick up two copies. We don't know if they're on the same chromosome or not. Occasionally, they may be on the same chromosome, and an individual with two copies of SMN1 is going to be a carrier, because that other chromosome has zero copies on it, and they can transmit that chromosome. So this is now added in there to say, if we're detecting this, there's at least a higher chance that the two SMN1 genes we're identifying may be on the same chromosome. However, it is ethnic-specific, and you can do a Bayes analysis with the frequency and knowing how often these are to get a post-test carrier risk if you have one of these variants. I took this off of ARUP's site. In case people didn't know, I used to work at ARUP laboratories until about 10 months ago, so most of this is coming from my experience. Here's the newborn screening. So we need it inexpensive, but a high sensitivity and specificity, and then you go into the confirmatory test, which is SMN1 and SMN2, and then they can be approved for Spinraza. It's an antisense oligonucleotide. It turns SMN2 to a full-length mRNA acting like SMN1. This is an example of what a newborn screen could be. So these are primers that amplify up into the variant, and so you're getting a very, very short product, and you do a melting curve, and basically you're getting this as normal, and this is with a homozygous deletion is going to be shifted a little bit lower because this is only going to have the SMN2 variant on it. This shows you that you can actually identify which are SMN2s and which are SMN1s, but you really don't want this for carrier testing. You just want a quick call to say this is affected. You really don't want to know carrier status, so you just kind of ignore all of that and maybe call it there. Here, one other point is that the red, we can't tell by something like this if it's two copies and two copies or a one copy and one copy, so we really can't tell variant unless we have another gene as a control gene for copy number. Okay, quickly going on. The other two are dominant inheritance mainly, and just a reminder that with dominant inheritance, we do look more for de novo mutations, potentially reduced penetrance, more variability, and the first one I'm going to talk about is Charcot-Marie-Tooth hereditary neuropathy. So this is complicated. It's complex. It's a group of disorders with many types and subtypes. That's my summary for you. If you go into gene reviews or anything else, this is going to be very, very complex, so I've tried to simplify it but give you enough detail to make sense out of it. So hereditary neuropathy with a liability to pressure palsies is one type, and then different types of motor and or sensory neuropathies or muscular atrophies. Usually onset is in the first or third decade, but it depends. The one that we test for first is a PMP22 deletion duplication. Different methods, southern blot, PCR, over known breakpoints. A microarray or MLPA will do it. But the first tier with PMP22 deletion duplication, a duplication is an autosomal dominant and about a third are de novo, and that is Charcot-Marie-Tooth type 1. A full gene deletion actually is a different disorder. That's hereditary neuropathy with liability to pressure palsies. So this one gene, depending on whether it's duplicated or deleted, is going to have a different phenotype with it. Now it becomes more complex when you want to go into the rest of CMT, and this is done by gene panels. Looking for sequence variants. Also could be looking for deletion duplications of other genes. I've only done CMT type 1, showing some of the more common genes, but there's different subtypes and the clinical sensitivity of each one of this. So this gene accounts for 6% to 10%, et cetera. So you can see that this becomes complex very quickly. There's over 80 genes associated with Charcot-Marie-Tooth. So this is where panels become helpful, and the inheritance can be dominant or recessive. So believe it or not, if you look into OMIM, sometimes it's autosomal dominant, sometimes it's recessive, and sometimes you'll get autosomal dominant or autosomal recessive, meaning that there have actually been two types described or X-linked. Any questions on Charcot-Marie-Tooth? I've really given you the highlights. No BMP22, no deletion duplication, and interpreted they're going to give you the different diseases. Okay, and if I can do this in just a few minutes, we'll be back on schedule. So neurofibromatosis. Again, there's a diagnostic criteria, so this can be diagnosed without a molecular confirmation. If you have two of the following, greater than six cafe-au-lait macules, fretling, neurofibromas, leash nodules, optic gliomas, et cetera, first degree relative with NF. There are some other findings, sometimes learning disabilities, skeletal dysplasias, some overgrowth, and then you have the complications to this disease, particularly the neurofibromas, and then a risk for malignant peripheral nurse sheath tumors. It is dominant, about 300 to 400 worldwide. It's fully penetrant by adulthood, but not so much in childhood. The de novo variants are up to about 50%, and then you can also have large deletions of NF1 that are associated with the malignant phenotypes or other severe phenotypes. So molecular testing, multi-step. So in this case, we do sequence analysis first, right? And then we do deletion-duplication analysis. Sometimes it is so large you may want to go to a cytogenomic microarray for a cytogenomic analysis. Phenotype-genotype correlation, there's a little bit. So mainly it's with NF1 whole gene deletion. They are larger numbers of the cutaneous neurofibromatosis, more frequent and more severe cognitive abnormalities, more growth symptoms, more dysmorphic features. There's a three-base N-frame deletion that doesn't really have the cutaneous or a specific missense variance of this one specific codon, just to let you aware that there's a few little hot spot points in this gene. I just wanted to go over one case with you with this. So this is the clinical background. It's a one-year-old male with three cafe au lait macules and no relevant family history. What is the appropriate or recommended testing? And why would we do it? And what are your thoughts about this? What? Have them come back when? Okay. So the first question is, is this testing premature? Okay. They certainly don't have more than six cafe au lait spots. There is a recommendation that says if you're seeing kind of a high number of cafe au lait spots but it didn't put a number on it in children, you may want to do this anyway. However, this came from a geneticist who had a really good knowledge of NF. Do you question it or do you run it as a laboratory? What? Run it. We ran it. We really didn't stop and ask questions because we recognize the physician and we did it anyway. Sometimes we do question, especially if it's the wrong test, okay? So if you look at this and say, oh, my goodness, they want to start sequencing SMN1 and they haven't done a deletion test, then you stop them and say, you know, we really recommend deletion testing. You talk with them. Why are you doing sequencing? Have you done deletion testing? You know, we really recommend this first, and not just we, but it is recommended in the field. So we can stop it. But on the other hand, if physicians push back and say, I know what I'm doing, I want this anyway, you do what I tell you, we kind of stop arguing with them. Or we don't argue with them. We try to maintain professional. So they ordered full gene sequencing and deletion duplication. Deletion duplication, two copies were detected. It wasn't a full gene. I mean, it wasn't deleted or duplicated. We found a missense variant. It was in all transcripts. It was we did have parents. We did a family study. It was not present in either parent. We confirmed paternity and maternity by STRs. What do you think? Tomorrow you get more of an interpretation of sequence variants. But, you know, overall the evidence is that it wasn't in population databases. It was zero. Computational predictions are saying it's probably damaging or deleterious. So there's pretty good computational prediction, but that's still just kind of supporting evidence and not strong evidence. There's no predicted splicing effect with it. Family study demonstrated de novo with parentage confirmed. That's actually strong evidence. So strong de novo, moderate is that it's very, very rare, and one supporting and that the computational evidence was supporting. So we called it likely pathogenic. Comments? Questions? What was that? Yeah. So why were they bringing this child in in the first place to a geneticist? Yeah. I will imagine we didn't get all the information from the clinician. Okay. Any other questions? What would you recommend for this? Anything more? Follow up on the patient? Okay. You know, this may just be me, but I'm careful to not recommend clinical management as a laboratorian. I'm not sure that I have the expertise to recommend clinical management, but, boy, do I have the expertise to recommend other laboratory testing, right? So I really make sure I do that. Now, I do sometimes put a general in, you know, this patient needs to be, you know, followed by a specialty clinic or something like that. But because it was a de novo and there is a small but still a risk for gonadal mosaicism in the parent, that any testing for other offspring, particularly any future fetal testing, we could look for that exact mutation just to confirm it's not being seen again because of gonadal mosaicism. Testing method depends on the pathogenic mutation spectrum. Yes. Sorry. Go back. The gene what? For legis? If it was negative? For this? Typically I wouldn't, okay? I mean, yes, there's always a possibility that there's more than that. So you're doing, so you're doing both and both together all the time? Okay, yeah, I haven't done it that way. That's as much as what I can say. I guess my big conclusion is know your genes, okay? Every gene has a different story. That's very difficult in exome and genomes, but particularly for these single gene disorders, learn what's special and what you need to do. These are some of the common ones that you're going to come up with. Okay, I can probably take another question because I started a little bit late, but I've got you up. No questions? Then I think we can, oh, yes? When you were at ARUP, how many gene testings were you doing? I mean, I'm coming from a very small academic lab and I know what we do, and I just can't imagine how you can. How you manage it all? Also how you can do all this testing, too. I mean, I've worked in large reference labs, but still. So basically, we had four molecular genetic laboratories. One that was high throughput, targeted, you know, variance factor 5, factor 2, CF was done in there. We had our fragment analysis, which did more of the sizing, Fragile X, Huntington, STR analysis, so that was a second laboratory. We had our Sanger sequencing laboratory, and we had our next-gen laboratory. So each laboratory had a staff of about 15 to 20 people. They tried to keep it about that because that's what they felt a supervisor could manage, and then we floated around to the different laboratories. But as one medical director, we had five medical directors. We had more than that of genetic counselors helping to manage. We had more than that of sequence clinical variants scientists helping get us all of this, and so it became a huge team effort. Supervisors and managers that kept the workflow going and did all of the cap-related regulatory things, so it was a huge team effort. We had, I basically say, 500 different tests of genes and gene combinations, but that would mean that, you know, if we had cystic fibrosis, targeted, you know, a panel, sequencing, deletion, testing, sequencing and deletion, or reflex from panel to sequencing to deletion. So that's where we get the 500 combinations to manage. We probably had maybe several hundred genes in there. It was fun, but we grew that. I started there with Factor V Leiden and ApoE and a bone marrow transplant monitoring by the NTRs, so this was all grown. It wasn't starting off. Okay, and then who is next? Cindy, are you up?

neurogenetic testing

muscular dystrophy

spinal muscular atrophy

Charcot-Marie-Tooth hereditary neuropathy

genetic basis

testing methods

phenotype-genotype correlations

sequence variants

laboratory setting