 I want to thank Terry and the organizer for invitation to participate in this very exciting meeting. My interest has been in congenital heart disease for some time and Cricket gave a beautiful presentation earlier and she really introduced a lot of the background. So I'm going to be very brief. Congenital heart disease, as you know, is one of the most common birth effects. It's characterized by abnormalities in the cardiovascular structures. In particular, human and air breathing animals in general have four chamber hearts with separate pulmonary and systemic circulation. And so you have a right atrium, right ventricle, pumping the oxygenary blood through the pulmonary artery to the lung where it gets oxygenated. It returns to the left side of the heart into the right, into the left atrium, left ventricle, out the aorta and then systemically bringing oxygenary blood to the rest of the body. So there's this very distinct right-left asymmetry and four chamber anatomy that's really critical for survival of air breathing animals. And it's disruption of this four chamber anatomy that actually underlies congenital heart disease. As Cricket mentioned, advances in surgical palliation now allows most congenital heart disease patients to survive their structural heart defects. And in fact, many of these patients are living into adulthood with more adult congenital heart disease now than their children or infants born with congenital heart disease. What has been found over time, and again Cricket mentioned that patients with some of the same structural heart defects can have very different outcome. A particular note, for example, are patients with hypoplastic left heart syndrome. They have a very stenotic aorta with hypoplastic left ventricle and a tritic or stenotic mitral valve. So the whole left side of the heart is very hypoplastic. Patients with HLHS can have variable outcomes. Some after surgical palliation could live a relatively normal life and others undergo heart failure. Studies by the PHN network have shown that in fact, it's what's most important for the long term outcome of these patients are patient intrinsic factors and not surgical parameters. And so suggests that in fact, something intrinsic to the patient is actually determining outcome. And so all interest in the possibility of exploring whether genetic factors might be a contributing factor to possibly the differential outcome in patients with congenital heart disease. And of course the first step is to deciphering what are the genes that may contribute to congenital heart disease. And so that's what I'm gonna focus on. So we have undertaken a large-scale mutagenesis screen to look at genes that can cause congenital heart disease using the mouse's own model system. And the reason for that is that mice have the same four chamber cardiac anatomy as humans, so that's obviously important because it's the substrates of congenital heart disease. And also within bread mice, we can avoid the problem of genetic heterogeneity that you see in human studies. So it's really an ideal model system for studying the genetics of congenital heart disease. So we undertook a systems genetic approach, as I said, with a large-scale four-genetic screen with chemical mutagenesis with ethonitrocelluria. This introduces random mutations in the genome and allows us a way to interrogate in a non-gene bias way for genes that can contribute to congenital heart disease. So we're doing a phenotype-driven screen and the goal is to identify genes and pathways that may drive congenital heart disease pathogenesis. By driving this screen with phenotype, we're hoping the collection, the totality of genes may give us some insight what are the main pathways that are contributing to the pathogenesis congenital heart disease. And of course, we're also hoping that the totality of data we collect may also give us some insights into the genomic context for disease pathogenesis. So this is just some ultrasound images to show you the way we do the phenotyping is by in-utero-fetal ultrasound imaging. This is a whole conceptus that you can see, and that's the frontal view. You can rotate the transducer and you can see in the sagittal view the same animal, vertebral column, the heart beating way, and here's the head of the animal. And so this very relatively low resolution ultrasound will allow you to locate where the fetuses are and with the in-utero-fetal screen, we don't have to worry about fetal demise because we can actually track them horizontally over time. And so using more high resolution imaging with VVOL 2100, we can actually look up and get a lot of details and structures of the heart. So using four different imaging modality, we can actually collect very specific information about heart structure and function pretty much is what you would do for human fetuses or even human adult cardiac phenotyping. And so with fetal ultrasound, we are able to not only be very high throughput because it's non-invasive, but also have very high detection sensitivity and specificity because this imaging modality specifically developed to phenotype for cardiac anomalies. So using this non-invasive phenotyping strategy over a five-year period of screening, we've scanned 100,000 fetuses. This is a recessive screen, so it's a G2 by G1 back cross, so they come from over 3,000 pedigree. And out of the 100,000 fetuses, about 3,000 show evidence of cardiovascular defects. Obviously, only a subset of these will have structural heart disease. But by doing the fetal ultrasound imaging, we can go from the 100,000 to the 3,000 relatively efficiently. And we've been able to recover more than 300 mutant mouse lines with a wide range of congenital heart defect phenotypes. They're just grossly summarized here. I'm not gonna go through that with you, but suffice to say most of the congenital heart disease observed clinically have been recovered in our screen. One of the surprising things was that we found that 25% of the congenital heart disease mutant lines actually exhibit left-right pattern defects. If you remember to the early part of my introduction, I showed you the left, the cardiovascular system is very left-right asymmetric. And that asymmetry is actually really important for establishing systemic pulmonary circulation. So maybe you shouldn't be surprised that mutations that affect lateral defects actually give you some of the worst and most complex congenital heart disease. One of the, also one of the other interesting observations from the screen is that when you have a mutation that cause left-right pattern defects and congenital heart disease, that most of them will present with three phenotypes. So they can have cytosolidus, meaning the visceral organ cytos is completely normal. Cytosynversus, where it's reversed, a merisymmetric reverse. So for example, the heart's pointing to the right, but the stomach is on the left side, sorry, on the right side as well, which is the merisymmetric of normal, or you can have heterotaxi where it's randomized. So this animal has the heart pointing to the right, but actually the stomach is on the left. And so when you have heterotaxi, that's actually when you have congenital heart disease. But animals that have cytosolidus or cytosynversus are completely normal. I wanted to bring this up because this suggests that, well, we know that a single mutation can give you three distinct phenotype, only the heterotaxi mutants with congenital heart disease. So when we're doing exome sequencing or whole genome sequencing, and we're using sibling controls, I just want to point out that you may actually be filtering out mutations that could cause congenital heart disease. So for example, in the exact database, I think that that's something we should really keep in mind as a potential complication. So all of the mutant lines are recovered. We crowd preserve the sperm so that they're available from Jackson Laboratory, but we also actually provide very detailed curation of their phenotype. There's also deposit of the mouse genome informatic database. So this is an example of some of the images that you might see from one of these pages of a mutant. We actually have added several hundred mouse phenotype ontology terms for congenital heart disease. And we also use the Boston phylocodes so that we can correlate the mouse congenital disease with the phenotype with the human clinical terms so that they're cross-referenced. And we also provide, I forgot to, yes, we also provide a lot of images and Cine clips. So if you go to MGI, you can get a lot of detail information about the phenotype of these mutant lines. So of course, it's all about mutation recovery, not just getting interesting phenotypes. And we're able to do this by whole exome sequencing because we do our screen in the context of completely inbred C57 black six mice. So we can simply do exome sequencing, then compare any changes relative to the reference genome. And so at the end of our screen, we've generated over 12,000 mutations and over 7,000 genes. If you assume 24,000 genes, it would suggest that we're at 30% saturation. And of the 1,500 homozygous mutations, we see that 147, in fact, are pathogenic or we're able to show that they're pathogenic. Two thirds of them are missense mutation and the remainder are split between nonsense mutation and splicing defect mutations. And these reside in 98 genes. All of the mutations recovered, the more than 12,000 mutations are searchable in a mouse model organism search page in the Bench-DeBassenet website. And remembering that we've crowd preserved all the sperm, all of these alleles are actually available because you can regenerate them from reanimating the sperm. Of the 98 genes we recovered, 47 are novel genes, novel meaning not previously known to cause congenital disease. And we also have 23 genes with multiple leels. And using that screen metrics and a statistical modeling approach known as unseen species method, we can get an estimate of the total number of congenital heart disease gene in the mouse genome. So this includes looking at the total number of congenital disease genes recovered, the number of genes with one mutation and the total number of mutations recovered all together. And so this estimate is 272 genes and remembering this in the context of a recessive screen. So this will suggest that our screen is at about 35% saturation, remembering that we've recovered 98 genes. So this number 35% is remarkably close to the 30%. We came up with by just looking at the exome sequencing metrics. So taking a close look at the homozygonal mutations in the mutant lines where we recovered the pathogenic mutation, we see that there are 151 of these homozygonal mutations. We would expect that 30% of these would be embryonic lethal based on the COMP studies. Of these 151, 108 have been curated with known knock on mouse phenotype. And we see that of the 108, 104 or 96% are viable to weaning. That's not surprising because again, we're screening mice in mid-gestation, so this is selecting for genes that are not gonna cause early embryonic lethality. Surprisingly, we also recover mutations for null mutations in four genes that are not known to cause early embryonic lethality, but we actually get them to survive up to near term. So this suggests the notion of genetic resiliency which is recently presented in an interesting paper in Nature Biotechnology. So what do we recover in the totality of conjunct disease genes? We find that there is an enrichment for cilia-related mutations. 50 of the 98 are cilia-related with another 21 involve in cilia-transduced cell signaling. We also see a number of endocytic vesicular trafficking genes. Overall, this would suggest that the disturbance of cilia-cilia-related function may play an important role in the pathogenesis of congenital heart disease. This is just a diagram to show you some of the cilia genes that were recovered. I'm not gonna go through this, but I just wanna point out that we obviously recovered some of the cilia genes that are involved in human disease, including genes involved in multi-cilia function shown here. They can cause primary cilia dyskinesia, but also genes involved in ciliopathies that are thought largely to involve primary cilia, including genes that cause uber syndrome, mechogruber syndrome, nephromythesis. And many of these genes you can see actually are found in the cilia transition zone, which is a very important region that gates trafficking of proteins in and out of the cilia. We also found many cilia-transduced cell signaling genes, 21 in total, including those involved in wind signaling, in hedgehog signaling, PGF signaling, and TGF beta signaling. And so the question is, do these pathways and mutations really have any relevance to human congenital heart disease? So we borrowed some data from the pediatric cardiac genomics consortium that Cricut mentioned earlier. This is data actually from the earlier study that was published in 2013. Out of 27 de novo variants that were recovered, we found that 11 of them were in pathways identified by a mouse congenital heart disease screen, including genes involved in TGF beta signaling, wind signaling, hedgehog signaling, cilium-related, and endocytic trafficking. Again, to reverberate something that Cricut mentioned, we found that actually many of these genes are involved and also have a role to play in exon guidance in neurogenesis and synaptic transmission. Here's just a subset of the genes recovered. And actually now with the 98 genes, if we put it through pathway analysis, we see that one of the top pathways recovered indeed is involved in exon guidance in neurogenesis. So again, suggesting the sharing of developmental pathways and heart development and development of the brain and the nervous system, providing an explanation for the poor neural developmental outcome often seen with congenital heart disease. Another interesting finding that we made was that many of the pathogenic mutations recovered actually code for interacting proteins, that is proteins that are direct interacting partners. So here are just some examples. So we have a mutation in one animal, that's ang6 that causes a disease, a different animal in neck eight, and we know both of these proteins are direct interacting partners, but these were recovered in separate animal, completely independent of each other. And this is repeated many, many times. For the transition zone complex, there are too many proteins to list. And so the question is whether in fact, genes that are involved in congenital disease pathogenesis might actually be part of an interneptal network. And so to look at that question, we took the congenital disease gene and created the HPRD biogrid database that has information on protein-protein interactions and generated interactome network as shown here. And we see that the shortest distance between nodes, which are the congenital disease genes in red, has 4.7 edges. If you do the same kind of interactome assembly using random gene sets and you simulate that 10,000 times, we get 14.9 edges, suggesting that this congenital disease gene network actually is a functional significance. And so this led to this notion that maybe the interactome network itself may provide a genomic context for congenital disease pathogenesis and that this may explain the complex genetics of congenital disease, which is really more relevant to human disease than recessive mutations. And so can we provide some experimental evidence to support that? So with that in mind, we looked at ANG-6 and NEC-8 in collaboration with Jaga Shell. We previously showed that ANG-6 actually is required to activate NEC-8. NEC-8 is a kinase. It's actually the only kinase that's been identified in the cilia. And so in the presence of ANG-6, NEC-8, kinase is activated. And so the question is, we have this mutation in ANG-6 and a mutation in NEC-8. Can these two genes interact in an epistatic fashion to cause disease? To look at that question, we intercross heterozygous, ANG-6, heterozygous, NEC-8. Each one by themselves in heterozygosity has no disease phenotype. With the intercrosses, you get four genotype outcome. Of course, what's of interest is the double heterozygous. You would expect that a 25% ratio. We can see there's a depression. We're harvesting these in utero, so they're late gestation embryos. And we can see that actually we're losing some of them much earlier. And actually out of the 27 embryos, we did recover. 17 of them show mutant phenotypes that are seen only in the homozygous ANG-6 or homozygous NEC-8 mutants. So it shows that diagenic interaction, double heterozygosity, can actually give you disease phenotype similar to what you would see in the homozygous adult animals. Suggesting this concept of multigenic interactions in an interactome network may really be relevant for thinking about human congenital disease. Finally, I just wanted to show very quickly that we've also been very interested in hypoplasic lab heart syndrome, which Cricket also mentioned earlier. It was surprising. This is one of the rarest phenotype we've covered on our screen. We recovered eight mutant lines with HLHS. Surprisingly, exome sequencing analysis show that these eight mutants have no genes in common. I'm suggesting this notion that they have a multigenic etiology. We've been able to validate that for one particular line where we can show that there are two genes required to generate HLHS. So we believe that this is a disease phenotype that is intrinsically multigenic in etiology. And we believe that there is a couple of other congenital disease phenotype in that same category. So to close, I just want to leave you with this thought that using system genetics with mutagenesis may provide a segue for really interrogating the complex genetics of human congiart disease. So not only can we use forward genetics to recover and gain insight into recessive mutations that are in a Mendelian genetic model of disease, but that these may lead us to insights on the complex genetics of disease because I believe the same genes that are in these Mendelian model can also contribute to more complex genetics of disease. And moreover, we think that using this system genetic approach may give us some insight in the genomic context of disease pathogenesis so that looking at the totality of mutations recovered, we may actually gain some insight in genetic resiliency. And one thing I didn't even talk about is whether there's evidence for protective versus pathogenic alleles, which I believe our screen may have some evidence for, and also this concept of penetrance, which we touch upon a number of times. And finally, I want to suggest that maybe there's real value possibly provided by generating immunogenesis database. There are many, many labs that are doing screens. The collection of that data may perhaps allow another means to query sequence variants for possible functional significance. And again, this is something that obviously will require some resource to collate. And so in closing then regarding the question of animal modeling of human disease, I would like to propose that in selecting animal models, you obviously need to make sure that there is similar anatomy and physiology in your animal model to the human disease under study. And so for congenital heart disease, you need a four chamber heart. The availability of inbred strains is also important in the context of genetic analysis and mice is uniquely suited, given that we have completely inbred strains. Phenotype ontology is also very important. It's really critical that the phenotype ontology used to describe phenotypes in animal models parallel to human phenotype ontology. And I think this is something that needs to expand with different animal model system under use. And finally, there needs to be a way to quickly disseminate the phenotype genotype data in public databases so clinicians and other scientists can readily access that. For us, Jackson Lab has been wonderful in providing MGI as a context in which we can do that. And then finally, I think Hao Yi Wang may have mentioned the use of CRISPR-Cas. Ultimately with animal model, can we then use CRISPR-Cas as a quick way to do validation of human sequence variants? And so with that, I'd like to thank a lot of people that contributed to the work and support from Heartland and Blood. I did have a lot of clinical collaborations. I didn't really have time to show the data, but we provide a lot of mouse models for a number of papers that have been published on different human diseases. So with that, I thank you for your attention. Thank you. I have a quick question for you. And then I think we'll move to a discussion. So your heterozygotes are fascinating. So you didn't see complete penetrance with the two heterozygotes. So have you looked at these animals in more detail and let them live longer to see what happens? So the double heterozygotes animals, if they are cytosolidus, they're perfectly fine. If they're cytosinversus, they're perfectly fine. But it's the one with heterotaxi. They always have complex congenital disease, and those expire. But the ones with cytosinversus are perfectly fine, just like the homozygotanus. So this is a line where we see laterality phenotypes. And we actually have another line that does not have left-right pattern defects that we also have seen this diagenic interactions. So I think that this is, you know, something that I think will be important that we can explore experimentally. We're trying to figure out a way to do this more systematically based on the protein interactome. So, but, yeah, I don't know if that answers your question. Have you crossed any of the other combinations? So you just looked at the one set of genes. Have you looked at any of the other combinations to see what happens as heterozygotes? So, yes, we have another combination that we've done that we also see interactions. And the phenotype in that case is actually milder than in either of the parental homozygotes. But we still see the congiard disease penetrant. Some of the extra cardiac phenotypes seem to be milder or not present. But the cardiac phenotype is still persists. Okay, any specific questions for Cecilia? Or open it for, yeah, quick it. Cecilia, that was great. I was just curious about your genetic resiliency. Did those mice not exhibit the phenotype or did they have a phenotype in survival? They have congiard disease that's related to another gene that we were tracking. But the severe embryonic lethality phenotype known for the knockout is obviously not observed because we're getting these mice to term. So in other words, one example is like LRP1B, it's one of the homozygogenes we found in homozygosity. The knockout mouse actually dies around nine and a half, 10 and a half days gestation. So our mutants are being examined near term. So they're coming to term. So basically that knockout, that homozygote knockout really has been rescued, if you will, by other mutations in that line. And I'm not saying that it's necessary the pathogenic gene that we recovered, but suffice it to say that the genomic context of that animal has rescued that null phenotype. Yeah, so we have the exome data just from the one animal. And so yes, there are some suggestion of other genes in endocytic trafficking that actually may provide some recovery of function. But obviously, we don't have experimental data, it's just argument based on the exome sequencing data. Callum, Cecilia, I was gonna ask you if you'd looked at cellular phenotypes across clutches without, sorry, litters, across litters without, and correlated them with cardiac phenotypes. For example, a ciliary... Right, right, so yes, so we've looked at ciliation efficiency, percent ciliation, and asking, is it consistent between animals? Yes, they are consistent between animals and right now what we're trying to do is actually look at kinase activity. So in the double headers, I get what happens to the kinase activity and the interaction between angst six and egg eight. So we're trying to do some biochemistry to really address that question. And we also, of course, have animals with tissues that we can analyze as well. So we're looking at that. And these two mutations cause nephronithesis. And so we actually can also see that they have a cystic kidney phenotype, the double heads. But I think they're milder, although we haven't looked so carefully that we've quantitated, and I can say it's truly milder, but they definitely have nephronithesis just like the homozygote, but it seems like it takes longer for them to get out to that more severe state. Other questions? Any comments for Peter? I wonder if the terms reduced penetrance and genetic resiliency shouldn't just be dropped because they're both euphemisms for oligogenic inheritance and actually imply things about the situation which might not be true. What you're saying is penetrance, your focus is on one mutation and you're saying, well, this mutation has the characteristic that it doesn't always cause disease. Well, actually the situation might just be that there's one, two, three, four other genes that determine together whether disease comes out and your focus on this one gene is just a historical accident. That's what I mean, it's not really a good term. Sure, no, I understand, but what would be the alternative? Just that these are polygenic traits that are complex. I mean, that seems like a long way of saying reduced penetrance. Well, I think it's a more honest way of saying it. Correct it. But it's not necessarily genotype. It could be dietary. It could be other things. So I think we can't just assume everything is true. Well, especially with left, right, there's a clear stochastic component of fright. Exposures matter. We know viruses cause disease.