financial disclosures, two reports, and the next slide shows the planning committee and their financial disclosures, which are indicated. This session is approved for one continuing medical education credit, and to obtain credit, just complete the evaluation form at the end of this session. The next slide shows the overall learning objectives for the Genome Case Conferences in general, and today our session learning objectives are listed here, but Dr. Kruska will go through them as he begins the case conference. Hello, everyone. It's great to be here and talk about our research for holoprosencephaly. Before we start, I'm going to just tell you my personal connection to holoprosencephaly. I saw my first holoprosencephaly patients in 2014 at the HOPE Conference, which is a family conference, and I talked to hundreds of families affected by holoprosencephaly, and what I learned were two things that I really could boil down. The first thing was that the families wanted to know why their child had holoprosencephaly and if their future children would have it, and the second question was how to deal with many of the sequelae of holoprosencephaly, such as diabetes, insipidus, and seizures, which I'll talk about. In this talk that Don and I are doing really addresses that first question, why do people get holoprosencephaly, and will their future children be affected? So here's the outline. First of all, we'll talk about holoprosencephaly very briefly. We'll go into a case report, which is really the meat of this talk, along with genetic counseling, and then we'll talk about some preliminary data that we have for you and our plans for future research in holoprosencephaly. So introducing holoprosencephaly, I'm going to talk briefly about the phenotype and molecular characteristics of holoprosencephaly. However, I put out a special issue last year on holoprosencephaly in the American Journal of Medical Genetics, Part C, alongside Ben Solomon and Max Munker, and this is a much more in-depth account of holoprosencephaly. So what is holoprosencephaly? It's failure of the forebrain to separate in early gestation, like all genetic diseases that have the spectrum, with the worst being alovar and the least severe being microform. Here you can see embryonic brains. This is from the Kyoto Collection. These are 3D reconstructions of histologic sections, and on column 1, which is A, C, and E, you'll see a normal-forming brain at Carnegie Stage 14 through 20. On the right, I draw your attention to B, D, and C. Notice that the prosencephalon does not divide into the telencephalon. The telencephalon goes on to be your cerebral hemispheres. I think this will be more clear when I show you some postnatal MRIs. These are axial MRI slices through the brain, and at the top, alovar, I want you to focus on that. There is no separation between the cerebral hemispheres. In the bottom left-hand corner is an axial MRI of one of my patients without holoprosencephaly here at the NIH. You can see the beautiful separation between the inner hemispheric fissure, which separates the two cerebral hemispheres. I apologize that I don't have a pointer, and I can't point this out to you, but you can see that it's a brain that has a nice, beautiful division, unlike the holoprosencephaly patients at the top of the screen. Not only does the brain not divide, the face does not divide completely. This is a CT scan, 3D reconstruction of one of our patients. This is a four-month-old, if you can believe it. You see that there's only one socket. The two sockets of the eye and the face failed to separate. On the theme of a spectrum, you can see that the facial characteristics of holoprosencephaly also are a spectrum. On the left is the most severe form. You have cyclopia, one eye, and then you have a nose structure above that, which we call a proboscis. If you go all the way to the right, we call that microform. That is the least severe form. If you look closely at her teeth, she has a single central incisor. Holoprosencephaly, as you can imagine, is a disease that is severe. It's not a surprise that it's more prevalent in the embryonic period. If you look at the left blue circle, again from the Kyoto collection, this was a study of elective terminations in Japan in the 1960s and 70s. They estimated that one in 250 conceptuses were affected with holoprosencephaly. Obviously, the birth prevalence is much less, more like one in 10,000 to one in 30,000. The first evidence that a single gene caused holoprosencephaly was this study from 1994 headed by Max Munker at the Children's Hospital of Philadelphia. On chromosome 7, they linked holoprosencephaly. Sonic Hedgehog, probably the most known cause of holoprosencephaly, the most known single gene cause, I should add, is on chromosome 7. This was a beautiful study. The limitations of studies like this is you need a three-generation pedigree to generate something called an LOD score, which is ancient history now. We'll show you later on in this talk next-generation sequencing that can pick up de novo variants. The Sonic Hedgehog pathway over the last two decades has been studied extensively. You'll see three red circles on this pathway, Sonic Hedgehog 6.2, 6.3, excuse me, and Zik 2, which are three of the common genes now that we test for. However, these three genes, and I added a fourth one, so the name of this slide is pre-exome sequencing project genetic etiology after excluding aneuploidy. After we exclude aneuploidy, so trisomy 13, something I didn't mention, is the most common cause of holoprosencephaly, but if you exclude aneuploidy, the genes I just discussed, and I added FGFR1, before we started our exome sequencing project that we're talking about today, only made up 14% of hundreds of cases that we have looked at here at the NIH. So 86% is unknown, and that really is the engine and driving force for our project. So we're going to start with a case report here, and this is a child, a boy with semi-lobar holoprosencephaly and pancreatic agenesis. He was born at 33 weeks gestation by C-section for intrauterine growth restriction. No surprise, because you see pancreatic agenesis in the title of this slide. Pancreatic agenesis is an important growth factor in the third trimester, intrauterine, and so no surprise that they went ahead and delivered this child by cesarean section. The child was diagnosed with semi-lobar holoprosencephaly on an ultrasound prenatally, confirmed later with an MRI. The child had neonatal diabetes, required insulin, and pancreatic exocrine deficiency treated with enzyme therapy. Now that is the definition right there, that third bullet point of pancreatic agenesis. Other medical issues, diabetes insipidus, which is a hypothalamic issue with diuretic hormone, and that's very common in holoprosencephaly, carrying loss. Global developmental delay, also extremely common. Almost everyone with holoprosencephaly has some type of delay. Family history, this is the family's first child. Pancreatics are healthy, and here is a picture of this beautiful baby boy. This was taken probably around 15 months or so, and you can notice there are some facial characteristics including intertemporal folds, he has a smooth philtrum that is long, and he has microtia bilaterally, CT scan of the head showed that he had some auricle obstacles deformities also. So our genetic workup was standard for this guy. We did sonic hedgehog 6-3-6-2-TGIF1, the traditional common genes known to cause holoprosencephaly. We did this in our CLIA lab here at NIH at the human development section, followed by research exome sequencing and Sanger sequencing confirmation of what we found. Exome sequencing was done, yes, on the HiSeq 2000, an ancient machine. We use NovaSeqs here now at NIH. For variant calling, we used the standard GATK pipeline, best practices. And I just want to point out that we encourage everyone to do copy number variations because obviously there can be deletions in an important gene responsible for holoprosencephaly. We did pull off copy number variation from the exomes using the software XHMM, and that basically just measures read depth on a large number of samples and uses statistics to figure out if there was a copy number variation. And so this is what we found with exome sequencing. Here is an IGV view, so IGV is a piece of software that is free from the Broad Institute, and it shows short read pile-ups, and here you can see all those little rectangles are roughly 100 to 120 base pair pile-ups that have been aligned to the reference genome. And you can see on the top is the proband, and he is heterozygous for this mutation of C to T transversion and CNOT1, which causes an arginine to sustain amino acid change at position 535. So this looks nice, good read depth. The parents don't have it, so this is de novo, he's heterozygous for this variant. Why is this variant initially interesting to us? Well, it's de novo. Statistically, we all have about 0.6 de novo variants in the exome. And here's the location on the CNOT protein, it's in the heat domain, this is a conserved domain that binds to other components of something called the CCR4 NOT complex, which I'll talk about briefly in a little bit. You can see in red or orange there that the arginine at this position at 535 is conserved from the human down to C. elegans. Another interesting point, and if you look on the NOMAD database, this is a very constrained gene, the PLI is 1, the observed to expected ratio is 0.01, that's very low, so humans do not tolerate loss of function in this variant. I'm not saying there is loss of function, but this is a metric that makes this call more interesting. And so what does CNOT1 do? Very briefly, it's part of the CCR4 NOT complex, which is responsible for, as you can see here, that little Pac-Man device eating up the poly-A tail, so it's a deadenylase, and this controls post-transcriptional regulation. One thing we did, we actually tried to make a zebrafish with this gene. We were not successful in eliciting the phenotype, but we did, this is Rob Lipinski's work out of the University of Wisconsin, these are 8.25 mouse embryos, the time that is very important for holoprotein cephaly in the mouse, and these are in situ hybridization reactions, and you can see we have a control called FOXA2, that's in the sonic hedgehog pathway, and that's a control, and kind of the two little, I guess it looks almost like two little ovals at the top, that's the neural fold, so you can see on the left, and CNOT1 is expressed in the neural fold, so this means that this gene is expressed in the right time and the right place for holoprotein cephaly, not proof that it causes holoprotein cephaly, but a little more evidence towards that, and here's from another study by Chen et al., and if you look at the top CNOT1, this is immunoblotting in the mouse brain, after embryonic day 13, CNOT1 is no longer expressed in the mouse brain, so I think this is important that it is expressed at the time of holoprotein cephaly. So we published this paper, we published this case and another case, so we found two cases in 134 trios, two cases of CNOT1, and the amazing thing is that they both had the same amino acid change, the arginine 535 sustained change, and so we had two exact cases, they both had semi-lobar holoprotein cephaly, and fortunately we were happy to see another group publish three patients, three with holoprotein cephaly and agenesis of the pancreas and they all had the same amino acid change, the arginine 535 sustained, so this was really remarkable and certainly supported our work to have five of these papers just by coincidence, well no they were not published by coincidence, but they were published together in the same journal issue of the American Journal of Human Genetics. The other group did some beautiful mouse work, remember our patients were heterozygous for this change, this is a mouse at embryonic day 14.5 on top is a wild type mouse, on the bottom is a homozygous mouse for this variant, and you can see the mouse has X encephaly, you can't really see it close enough in this slide, but they also have colobomas. This mouse worked very nice, supported our phenotype, and these are some beautiful pictures, that colorful diagram in the middle is a pancreas, and these are done from episcopic reconstructions and they're superimposed on a mouse embryo, and you can see way to the left that's the homozygous change and then to the right is the wild type, and if you notice to the left they're missing the green piece of the pancreas, that's the posterior pancreas, so this also supports our work, this is a decrease in pancreatic size of the mouse. They also did some expression studies, and interesting if you look all the way to the top left of this diagram there was a significant upregulation in Sonic Hedgehog, so that was really interesting, I think really we need to do some more research to figure out exactly what's going on with C-NOT1, so now I'm going to turn it over to Don Hadley, and I think equally important to the case study is really the genetic counseling with holoprosencephaly. Thank you, Paul. As Paul has described, holoprosencephaly may present with varying degrees of severity, it can be recognized at different times within one's life, it may or may not have causal explanations, and can be part of a broader syndrome, or can be present for generations within a family so mildly that it's not recognized, and finally and increasingly may allow survival into adulthood. This baffling array of possibilities can easily lead to uncertainty, confusion, depression, guilt, blame, and even fear as the information is presented to parents who most likely are not prepared to accept the new reality for their child. Within the provision of this complicated information, families often seek some level of hope as they learn about, adapt to, cope with, and make decisions about their child and their dramatically lives that lay before them. Today's webinar will provide a small sample of challenging issues encountered by families who receive a molecular result. For those who are interested in a broader view of the challenges encountered in education and counseling of families with holoprosencephaly, I encourage participants to read the article that's cited on this slide and also provided within the resources. Topics we'll touch on today include the education and counseling challenges presented to families with an autosomal dominant form of holoprosencephaly, including incomplete penetrance and phenotypic variability, parental mosaicism, and the array of counseling issues encountered by parents following molecular testing. Since I suspect that a large proportion of the audience are genetics professionals, I won't take time to define the terms penetrance and clinical variability. However, the next slide demonstrates these concepts for the four most common genes associated with HPE. This slide illustrates the proportion of individuals with a pathogenic variant in one of the four classic holoprosencephaly genes that present with structural brain anomaly, that's the dark bars on this slide, versus those without structural brain anomalies, the medium gray and the lighter gray bars to the right. Please note that those without structural brain anomalies consist of individuals with pathogenic variants and either microform findings, the medium gray bars, or who have apparently normal by standard clinical criteria, normal clinical exams. Those are the lighter gray bars. With that said, we suspect that many individuals described as being clinically unaffected may have unappreciated microform features. The clinical variability of holoprosencephaly within this family demonstrates the complex array of issues challenging families with inherited forms. The mother of the proban, Roman numeral 2-2, of the newborn girl, Roman numeral 3-1, exhibited far less anxiety at the birth of her daughter than most parents, having had her own personal experience with cleft lip and cleft palate. When we spoke with her about her daughter's cleft, she seemed confident in her ability to handle the problems presented by her daughter's clefts. However, as the diagnostic spectrum expanded to include the potential for significant intellectual delays and additional problems, medical problems, it brought a new level of challenges, including the potential that her child might die. The maternal grandfather, Roman numeral 1-1, expressed a sense of guilt for passing on the variant to both children and overwhelming sadness for causing his granddaughter to have such severe problems. The maternal uncle, Roman numeral 2-1, who did not yet have children, expressed how much he loved his niece and enjoyed playing with her. However, the thought of having his own child with needs as significant as hers brought pause to his consideration for having his own children. We suspect that at that point in time, his struggle reflected his perception for only the worst-case scenario for his children without consideration for a less severe phenotype, including that of his own, which was a retinal coloboma. As conversations evolved and included options for prenatal diagnosis, IVF, and preimplantation genetic diagnosis, he continued to express concerns over the lack of predictability of the clinical expression and became overwhelmed with the uncertainty associated with holoprosencephaly. While genetic and genomic technologies continue to grow and improve our abilities to identify genetic causes for increasing number of diseases and disorders, including holoprosencephaly, these technologies may also contribute to the uncertainty experienced by families. Uncertainty may arise from having little or incomplete information, imprecise information, or extremely complicated and detailed information. Likewise, uncertainty may arise from an inconclusive diagnosis, uncertain disease severity, and the lack of therapeutic or preventive measures. Feelings of uncertainty are often encountered by families following the diagnosis of a child with holoprosencephaly, even in the presence of an identified variant. Such uncertainty can leave parents feeling out of control, unable to plan, feeling unprepared to handle future events, which can lead to persistent emotional issues. Such challenges are complicated by the potential of parental mosaicism, and I'll ask Dr. Kruse to briefly present some recent published data on mosaicism in HPE. All right, thanks, Don. This is a really cool study that our group published earlier last year, and you'll see a red box there, and what that is is that is germline mosaicism in the parents. You can see it's the first column in that red box. It says F, 3%, so that's a mom with 3% germline mosaicism based on deep sequencing, so that'd be next-generation sequencing where we did a target capture of the gene of interest. In this case, it's FGFR1, so the sequencing depth was in the order of hundreds, four, five, 600. Then we also confirmed that with digital droplet PCR, which is just to the right of that, 3.7%, and it gets more impressive as you go down. The next row is the gene of interest, row is the maternal 1.54%, and then below that, it's even lower, 0.3%. So these are very low levels of mosaicism, so this reinforces what we all know in genetic counseling to always talk about germline mosaicism, even if you have a negative result in the parent, and apparently de novo finding, you still have to talk about the possibility of future children with holoprosencephaly. This slide lists some of the options for managing risk for future children and families with inherited forms of holoprosencephaly. These options are familiar to the clinical geneticists and genetic counselors, so I won't delve into specifics, however, I would like to emphasize the third and fourth bullets to highlight the potential that some parents facing recurrence may opt for no pre-pregnancy or prenatal interventions based upon their personal, their family, cultural, or even religious reasons. Such decisions might leave healthcare professionals feeling like they have no role in helping the family manage risk. However, no matter what choices are made, families may benefit from having a trusted professional and other families who have experienced holoprosencephaly to help them sort through the array of emotions and feelings that they'll encounter as the process unfolds with or without the use of prenatal and genetic technologies. Within the context of providing reproductive risk, families may experience a multitude of feelings, but not necessarily limited to those presented on this slide. I think what's very interesting is that there's little research that's available which investigates how families experience and manage diseases with highly variable, unpredictable, and uncertain phenotypic expression. Some of the research on this describes disorders with extreme variability as a condition without parameters and puts forth the premise that individuals confronted with disorders with such extreme variability do not have a model to draw upon in order to understand and manage their disorder. These phenomena are observed in many inherited diseases and disorders and may complicate the interpretation and the processing of the information and potentially induce feelings of guilt in family members less affected but identified as obligate carriers and raise anxiety in regard to reproductive decision-making and potentially affect how families communicate about the disease within their family. This is certainly an area within the provision of genetic counseling that deserves additional research in an effort to improve care. Due to the emotional traumatic nature of the diagnosis, the complicated, foreign, and uncertain nature of information being provided, follow-up sessions, whether in person or over the phone, may benefit the family as they process the information within the context of their own values, their own resources, and beliefs. The inclusion of specialists who focus on adaptation may benefit families as they process the information within the context of their own individual and family meeting. Likewise, and as Paul has already pointed out, opportunities to speak with families who face prenatal detection of holoprosencephaly, experience the birth of a child with HPE, or later experience the diagnosis following investigations for cognitive impairment may provide a resource unequal by healthcare providers. With that, I'll turn the presentation back over to Paul. Great, thanks, Don. So the case report that I presented before Don talked about counseling was part of our Next Generation Sequencing Project. So this data that I'm gonna present next is on data that we obtained from exome sequencing. So the first was CNOT1, we talked about that in detail in a case report. And this data comes from 322 probands, and here we have trios and we have singletons. And something I wanna point out about this graph, or I'm sorry, this table, is that the average age of our patients is very low. You can imagine why. Individuals with holoprosencephaly have a very shortened lifespan. And another thing I wanna point out here is that the most common cause, or the most common subtype of holoprosencephaly that we see here at the human development section is semi-lobar. Now obviously, we are subject to ascertainment bias, but those are most of the cases that we do see. So I call this preliminary data, exome sequencing preliminary data, because at the end of this talk, I'm gonna talk about genome sequencing, or whole genome sequencing, which is the next step, and we're really excited to share some news about that project. So preliminary data, or what I call exome sequencing, has been very fruitful so far. As you can see from this graph, the original genes, the Zik2, 6'3", Sonic Hedgehog, FGFR1, accounted for 14%, and I don't see that here, it just says percentage. It should say 14% there. And that exome sequencing has contributed 8% of genes that we're fairly confident cause holoprosencephaly. There's a few other ones that we're, the jury is still out. And so these genes include STAG2, which I'll talk about, SMC1A, RAD21, CNAT1, which we already talked about, KMT2D, and there should be one more gene here, and I'll talk about that. That's PP1R12A, we just published that this week in the American Journal of Human Genetics. Okay, let me go back. So the first finding I'm gonna talk about is cohesin-complex holoprosencephaly, and this is a really exciting finding for us. And just a quick review of the cohesin-complex. Most of us know the cohesin-complex as this ring of four molecules that keep sister chromatid together during mitosis. And we also know that it is associated with Cornelius DeLonga syndrome. Some of us may not know that cohesin-complex molecules also contribute to transcription control through enhancer-promotion interactions. And you can see in this cartoon, the cohesin loop made up of the four molecules, STAG1, STAG2 from the previous slide, SMC1A, SMC3, and RAD21. They kind of bring, as you can see, they loop around the DNA and bring the enhancer and promoter closer together. And so here is a chart of the first 15 patients we know about. Half of these are from our cohort. The other half are from colleagues of ours. And this is what we published, but I can tell you I get about one to two more patients a month since publishing this paper. So we're now up to about actually 30 patients with variants in the cohesin-complex genes. And this is really cool. If you look at the first, I guess, first 11, first 11 variants, first 11 patients, they're in STAG2 and SMC1A. And what's interesting is these two genes lie on the X chromosome. These are the first two X-linked holoprosencephaly-associated genes. These are all females. I assume this is embryonic lethal in the male. And also these are all loss-of-function variants. These are all truncating variants. And then the other, we have less of RAD21 and SMC as you go further down the table. And just like other slides I've shown previously in the introduction, there is a spectrum of holoprosencephaly associated with cohesinopathy. So this is from the same paper. You can see at the top, we have cyclopia. And then the next patient over, you can see, has facial clefting. And then some of these patients look average. And again, we did in situ hybridization in the mouse right at that critical time, embryonic day 8.5. And this was beautiful work done by Rob Lipinski at the University of Wisconsin. And the next variant, or next gene we published associated with holoprosencephaly, this was work done by Cedric Gonging here in the Human Development section. He found two variants in KMT2D. He was very careful not to call this Kabuki syndrome. As you can see from the photos of these two individuals, it's very hard to make the diagnosis phenotypically of Kabuki syndrome. And they also then have heart malformations. We are happy to see, though, soon after we publish that paper, that another group associated holoprosencephaly with Kabuki syndrome, confirming our findings. And then the last exome-related finding I'm gonna talk about was just published a few weeks ago, or a week ago. This is loss of function variants in a gene called PPP1R12A. Yes, that's a mouthful. This gene codes for a myosin phosphatase. And here's a table of the 12 patients we published. Only three of these patients were in our initial cohort. The other are from colleagues of ours. And there's really a spectrum here of brain malformations. I don't know if you can see this slide that well. And they also, interestingly enough, had urogenital malformations, too. So this is kind of an unusual finding. And again, Rob Lapinski's work, he shows that PP1R12A is expressed precisely at the right time in the mouse for holoprosencephaly. And this is work by Chad Vecina. He's also at the University of Wisconsin. And there's two columns there. One column are human embryos, or I should say two rows, human embryos. And the other is mouse embryos. And these are urogenital tracts. And this is immunostaining. And this shows that PP1R12A is expressed at the right time during urogenital formation. So that's our exome. But we figured that maybe we weren't capturing everything in our exomes, and we've gone on to genome sequencing, because remember when I introduced myself, one of our goals here at the human development section was to answer that first question of families affected by holoprosencephaly, what causes it, and will my future children have holoprosencephaly? And so this is kind of a scheme that we came up with prioritizing, because we collect a lot of samples, we see a lot of patients with holoprosencephaly, and we wanted to prioritize who we were going to sequence first using whole genome sequencing, and this is kind of a complicated graph, but the people we want to sequence first are trios. We're very interested in trios, because it makes the bioinformatics easier, and we're also interested in trios that have been sequenced previously and don't have an answer, because we are into gene discovery. So the first aim of this whole genome sequencing project was to interrogate the non-coding space of genomes and probands and their parents affected by holoprosencephaly, and as we talked about previously about enhancers and promoters, there's things in our genome such as enhancers that are not always covered by whole exome sequencing, or exome sequencing as people like to call it now, and this is really interesting. This just came out recently. This is the Sonic Hedgehog signaling pathway. They just found another enhancer, so there's seven known long-range enhancers associated with Sonic Hedgehog, so this really drives home the point that we really need to be interrogating enhancers, and whole genome sequencing obviously will do that. Specific aim number two. Specific aim number two was to interrogate areas of the genome not covered by whole exome sequencing, and this is from the NOMAD browser, and looking all the way to the right is exon one, then exon two, exon three. I apologize. Again, I don't have a pointer, but if you look at the light blue, those are exomes, and then the light green is genome, and you can see that on exon three, the coverage is poor with exome, so this, an advantage would be exome sequencing. To cover exon three of the Sonic Hedgehog gene, and this is an IGV view again, and this is short-read pileups, and if you look at the bottom of this slide, that is exon three of Sonic Hedgehog, and you can see at our sequencing center here at NIH, we do not cover exon three at all, so you can imagine we're missing a lot in one of the more important genes, Sonic Hedgehog. And so specific aim three is to create a genomic database for holoprosencephaly at NIH that will be used for reference by international researchers like some of you on this call and clinicians, and I can tell you we get emails and phone calls all the time about new genes, and it is really nice to look up in our database and give the clinician or research an answer of what we found in our research. So there's a lot of challenges with whole genome sequencing that we are working on, and when you do this type of work, you can imagine there's data storage issues, computational power issues. Fortunately, we have a BioWolf, we have a high-performing computer here at the National Institutes of Health, and that certainly makes it easy, and we also have a lot of resources here at NIH such as an animal core in-house scientist. And this is a slide from that issue that I showed you at the beginning, and it's from the special issue of the American Journal of Medical Genetics, and this was focusing on holoprosencephaly, and these are some commercial available testing for holoprosencephaly, and what I would like to see in the future with this research is for the column all the way to the right to change with some of these genes that we found in 2019 and we hope to find more in 2020. So here's the HOPE Conference. This is a family conference in Dallas. This occurs every two years. Don showed you that this is one of the resources for the patient, the HOPE Conference, and this is for us as researchers and physicians, this is a great way really to meet patients, meet their families, listen to their concerns. Here's the references that I used for this talk, and I just want to make some special acknowledgments here. One is the Carter Center for Brain Research and Holoprosencephaly and Related Brain Male Formations. This is at the Scottish Rite Hospital in Dallas, Texas, and this is a center that has really been important in our research, a driving force of finding patients for us, finding research participants for us, and also a referral place for us with patients with concerns. These guys really are the experts in taking care of folks with holoprosencephaly. The next organization I'd like to acknowledge is Families for HOPE, and this is really the premier patient organization, family patient organization in the United States that deals with holoprosencephaly, and I encourage all our listeners out there, if you have a family affected by holoprosencephaly, this is a wonderful organization with a lot of information for your family. And then last, I'd like to acknowledge the NIH Intramural Sequencing Center. They did all of our exome sequencing for the data that we presented here. All right, so it's time for questions and answers. We thank you for listening. So if you type your questions into the Q&A box in the top right corner of your screen, then we'll repeat the questions so everyone can hear. So we invite you to ask some questions. All right, so this is Paul Kruszka speaking again. The first question is that we received, okay, could one of the parents have had gonadal mosaicism for the CNOT1 mutation? So the caller is asking about the case report we presented, if you remember that child with agenesis of the pancreas, semi-lobar holoprosencephaly, and a missense mutation in the gene CNOT1. And so you saw the IGV pile up, right? And it was, what, about 60. So that would not be deep enough to necessarily cover gonadal mosaicism. Probably need to go into the hundreds, really, to cover low-level gonadal mosaicism or do droplet digital PCR. So this is a fantastic question. And really, should we be changing our practice for all genetic conditions in sequencing either deeper or doing digital droplet PCR? Right now, we don't have the resources to do that on every patient, but absolutely. This family could have had gonadal mosaicism for the CNOT1 mutation. What I didn't tell you in the presentation is they went on to have three more healthy children. So you can do your Bayesian analysis to figure out what the odds of gonadal mosaicism would be given they've had three healthy children now. So excellent question. That's the first question. Question number two. I'm gonna read the question here. It says, regarding pathogenic variants in cohesin genes, did you find, report facial dysmorphism? The patients in the pictures look like Cornelia DeLonga syndrome patients. Awesome, awesome question. Yes, absolutely. I'll tell you what. I think I talked too fast. I thought I was gonna run out of time, so I did not point that out. But yes, if you go back to that slide, some of the patients had arched eyebrows. And they did look, certainly the facial gestalt in a few of them did look like Cornelia DeLonga syndrome. So yes, very interesting. Excellent question. All right, so that's the Cornelia DeLonga. I'm gonna make another comment right now about question number two before I go to question three. If you remember for the cohesin apathy findings, one of the more common genes was the X-linked gene STAG2. And STAG2 has been reported in the literature in other papers. It was first reported by Dr. Martinez, and I think he's out of UCLA. And they've named the syndrome after him and his colleagues. So STAG2, I was surprised they didn't call that Cornelia DeLonga. They called it, they named it another syndrome. But yes, I do agree that some of the pictures absolutely look like Cornelia DeLonga. All right, going to question number three. It says, thank you for your talk. Can you speak to the reliability of copy number variant detection with exome sequencing and XHMM versus genome sequencing? Yes, another excellent question. It is much more accurate with genome sequencing. And I'm very happy that we're changing over to genome sequencing in our research here in the human development section. Yes, XHM is not completely sensitive or specific with copy number variations or getting copy number variants from whole exomes. The other thing is that you really to utilize exome sequencing with copy number variant detection, you have to have a large group because it's really statistical analysis. You're comparing exomes to the other exomes in your cohort, in your group. So you have to have quite a number of exomes to do that. You can't just do one exome and get a copy number variation whereas you can with genome sequencing, so yes. And I will tell you that we have published a number of reports of deletions in holoprosencephaly genes. So we highly recommend some type of copy number variation detection. Right now, probably a SNP chip is recommended with genome sequencing. Hopefully that will be enough in the future. All right, that's copy number variation. And then the last thing I will say, XHMM, we figured is better than nothing. Yes, we try to do SNP chips when we can, but it was really nice to have, to use that feature. All right, question number four. Heterozygous CNOT1 mouse have any phenotype? If not, how to explain the different mode of inheritance? Excellent question. I was wondering if someone was gonna say something about that. So no, the heterozygous, so we did not do the mouse studies. That was done by the DeFranco group, the other group that published on the exact same missense changes we did. And no, they did not have findings in the heterozygous mouse from what I can remember. Now, this is not uncommon in mice. We also see this in sonic hedgehog mice. The heterozygous sonic hedgehog mice does not have a severe phenotype, so you have to make a homozygous mouse. So same situation. Mice and people are not the same. All right, question number five. It says, sorry if I missed this, but do you know if CNOT1 acts via loss of protein function or is mice most likely to be gain of function? Well, great question, great question. I don't think in this particular case it's loss of function because if you go on the DDD website or the Decipher website, there are cases of CNOT1 loss of function variants, and we know of other CNOT1 loss of function variants from our colleagues, and they don't have holoprosencephaly or agenesis of the pancreas. So I don't think it's a loss of function, but I can't say for sure. I think it's, something else is going on. We actually don't know what it is, but I will refer you to the Decipher website. All right. So we have, looks like one more question here. The question is, is this being recorded? Could not join the webinar at the beginning since number of logged in participants exceeded the limit. Oh, great, we're popular, excellent. So yes, this has been, I was told from ACMG that this is being recorded and you should have access to this. All right. Next question, this is question seven. I'm gonna go ahead and read the question again and then give an answer. Just for my edification, when one is identifying gonadal mosaicism by deep sequencing, this is by sequencing sample from blood, not from gametes. When inferring the 9% gonadal mosaicism, can you use that when you are actually counseling about recurrent risk? I think the jury is still out for this. And absolutely, mosaicism is gonna be different in different tissues. And this brings out a good point. We are looking just at blood. And I don't know the answer to that. And as you saw, we had to do very deep sequencing either by exome sequencing with a capture kit or a digital droplet PCR, which obviously is 20,000 reactions. So it's just a very deep read. So yes, that's a good point. This is only blood. We did not identify any other tissues. All right, great. So that was question seven. It looks like we're actually a little bit ahead of time. We've been on the air for 54 minutes. So we still have a few more minutes if anyone has any more questions out there. All right, well, if there's no more questions, I'd like to thank Dr. Cooley for her presentation. I'd like to thank all of our listeners. And we have a few announcements to make before you guys tune out. So Don's gonna make a few announcements. Just like to invite the listeners to the next case conference. It's gonna be hosted by Baylor Clinical Center. And that's February 13th from one to 2 p.m. Eastern Standard Time. So please tune in to that case conference. We'd also like to make sure to invite the participants to the 2020 Annual Meeting, which will be held in San Antonio, Texas, March 17th through the 21st. And as was previously asked, recording of this seminar will be available in ACGME Genetics Academy at the link on the slide that's listed. And before you guys hang up, there was one more question. So thanks, Don, for going through the announcements. And the question is, for the HPE project, are you accepting samples from countries outside the USA? Absolutely we are. And please email me. Hopefully my email's somewhere here. You can Google my name. My email should come up. But yes, please contact me. I can talk about shipping samples overseas. We do it all the time. All right. Glad we squeezed that last question in. Thank you, everyone.

holoprosencephaly

genetic disorder

forebrain development

family questions

genetic variants

exome sequencing

copy number variant detection

whole genome sequencing

Carter Center for Brain Research