Scientist Stories: Hannah Mitchison, Expanding Ciliopathies

Thanks so much. It's a wonderful opportunity to tell people about the ciliopathy work that we do. And Enza Maria has given a fantastic introduction to cilia and to the primary ciliopathies. So I just flag up that we have been working in primary ciliopathies, but my talk is just going to be about the motile ciliopathies, which is also an expanding group. So I'm going to talk about the motile cilia and motile ciliopathies and the progress that's been made in molecular genetic basis of these diseases in the last 15 years. The first gene for PCD was cloned in 1999. So it's an expanding disease biology— disease category, and I'll speak something about the new biology that genetics has revealed, something about what we're starting to understand about the impact of individual genotype on the resulting disease phenotype, and a tiny bit about ideas for translation for therapy for these patients, because the motile ciliopathy patients suffer from a chronic, lifelong disorder of great morbidity. They have significantly less lethality than the primary ciliopathies, so they live a long time with a significant impairment to their quality of life. And they're— actually, it's not really very well studied about a reduction in the lifespan for motile ciliopathy patients, and there certainly is lethality, but it generally results from lung transplant and issues with respiratory distress more than sort of developmental lethality that Enzo Maria was speaking about. So we all should be familiar from basic biology with the beating cilia in the body, and we're very aware of the cilia that are beating in the upper and lower airways, and just showing here the respiratory epithelia, which is a mixture of the mucus-producing cells and ciliated epithelia in a quite complex structure that we can try and replicate in culture. We use air-liquid interface cultures of respiratory cilia that can be obtained by nasal brush brush biopsy. So if you place the nasal brush back to the nasal turbinate and make a good brushing, you can collect these ciliated epithelia. And you can see this very nice planar beating, which represents hundreds of cilia per cell, about 200 to 300 per cell. So I won't go into this, the difference between the motile and non-motile cilia, greatly, because we've we've heard a little bit about that already. But motile cilia do divide into multiciliated cell cilia and cilia that sit on their own. So the multiple— the multiciliated cells are in the airways, in the brain, pushing around the cerebrospinal fluid of the brain. They're in the female oviduct,. And the single motile cilia are in the embryonic node of— which Enzo Maria touched upon— they determine our left-right body plan, the laterality of the human body. And highly related are the sperm tails. So the flagella of the male gametes have an incredibly conserved structure with the motile cilia. And then we have these non-motile singleton cilia that account for the primary ciliopathies. So the cilia that are affected in PCD and motile ciliopathies— and I call it motile ciliopathies because we have a new class of motile cilia disease, which I'll talk about. So it's not just PCD on its own anymore. They're really on very specialized cells in the body. They're really not ubiquitous like the primary cilia. And these are just the regions that I was speaking about. And this is actually a sperm sample taken from a PC patient. They have very static cilia because of problems with the structure, the structural composition. And just to note also, because motile ciliopathies can be associated with retinitis pigmentosa, this very important connecting cilium in the photoreceptor of the eye. So these are really the regions of the body of interest for motile ciliopathies. And the laterality, this— the node that is a very transient structure that appears very early in development, about E7.5 in mice, is incredible and quite controversial, but it does seem to be true that single motile cilia that are beating in a helicopter fashion, so they beat in a leftward fashion, they're moving across important morphogens to the left side of the embryo that's sets up the left-right asymmetric body plan. And usually, in motile ciliopathy patients, they have a harmless complete mirror image reversal, which is called situs inversus totalis. So the lungs, the heart, the liver, these visceral organs all have a situs, and this causes no harm. The more concerning times are when there are isomerisms or heterotaxies, and it's generally a left isomerism in motile ciliopathies where you have no spleen or polysplenia and effects on the liver, and very importantly, congenital heart defects. These can cause very serious problems for these patients that require surgical correction and a much higher level of medical attention. So the primary ciliary dyskinesia group, so PCD, is the disease that you get when you have a problem with your motile cilia. It's a rare recessive disorder like all the other ciliopathies, and PCD CD, and the newer motile ciliopathies, collectively, they're thought to affect about 1 in 10,000 to 15,000 people. And this is the summary of the phenotypes. So, there's primarily a respiratory disorder due to problems with the mucociliary clearance in the lungs, and there are chronic infections and a diffuse congestive problem, a very classic and recognizable repetitive cough,, and lots of secretions that can interfere with hearing, with otitis media. There's reproductive consequences from not being able to move either the female gametes because of flagella cilia problems, or the male gametes because the sperm don't swim. There's the laterality problems that they have because of the lack of the flow at the embryonic node in this transient time during very earliest development. More often in the animal models, because they have a smaller brain, you see hydrocephalus, but this certainly is reported in patients, and they suffer from some more transient ventricle dilatations, and hydrocephalus seems to be particularly indicated with certain gene defects. And then there's one syndromic form that we know about, which is an excellent caused by mutations in the RPGR gene. So, this is showing the congenital heart defects, left isomerism. This isn't a person with a scisive versus— this is actually to show atelectasis, the collapse of the lung, and this very classic branching tree image that you see, which is this congestive and irreversible bronchiectasis that kills the lung, and the lung can't recover. Its function, and then the hydrocephalus from the cilia that line the brain ventricles. So we've been thinking about ciliopathies, and the motile cilia disorders do overlap, but they are pretty distinct, and in some ways are a little bit easier to think about with these various things. But we know that the primary ciliopathies are sometimes associated with similarly, similar-looking respiratory problems. So the exact connection and whether motile cilia are involved in primary ciliopathies as well, because motile cilia are also formed by intraflagellar transport and all the similar sorts of mechanisms, the jury's still out on that, the exact nature of the overlap. So just a couple of slides that a PCD, or Kartagener syndrome, was really first discovered by Dr. Kartagener in 1933 as this classic triad that I think medical students would classically learn about, the dextrocardia, so heart on the wrong side, bronchiectasis, and this terrible chronic respiratory sinusitis problems. In the '70s, another very important scientist for the motile ciliopathies, Bjorn Appelsalius, first linked ciliary defects to this condition. And in 1981, he proposed that structural defects of the cilia are at the base of the motile cilia problems. Then, as I said, in 1999, we got the first PCD gene, DNAI1. And now in 2016, I'm going to tell you about what we know about the 32 genes that have been isolated so far, and the fact that we think we can diagnose about 60 to 70% of cases based on those 32 genes. So PCD is very much an ultrastructural problem, and the classic motile cilia structure is here, the central tubules, 2 central tubules surrounded by 9 peripheral tubules with these little motor complexes. So the outer and inner dining arms are very crucial They have dynein motors that hydrolyze ATP to create the movement. And you have to have a very rigid structure of the axoneme, so there are nexin links, which are shown here, and the dynein arms in between these microtubules. It's a self-perpetuating forward and backward beat, and you have to have a pretty rigid structure to maintain that.— and speaking to your colleagues as well, these 200 cilia all beat in unison, and the precise way that that is laid down is not fully understood. So— and we have radial spoke proteins as well, which— these are just important for the genes I'm going to talk to you about. And just with regard to the photoreceptor, there was a nice review in Cilia in 2012 showing this amazingly conserved structure, so the transition zone, the axoneme, and the basal body nicely conserved. And when you do the cross-section, you can see that the 9 peripheral doublets, so a very similar organization. So I'm just going to present as a sort of an example, because it seems to illustrate a lot of the genetics that we know about PCD, is our— the way that we do diagnostics. In the UK and look at the genetics of the patients. So, patients are referred because they have a suitable clinical history. The first thing that happens is that they have their nitric oxide that's expelled in the nose measured. So, you can only do that from the age of 4 years and above because younger children are not able to hold the breath that's necessary for doing NO testing. So, younger children go straight to having a nasal brush biopsy, and the brush biopsy is subject to high-speed video imaging at the light microscope level. And at that point, many referrals are simply excluded. The cilia motility is totally normal, and that seems to be a pretty good diagnostic test. Then the rest are either clearly abnormal or have some slightly more or less well-defined ciliary motional defect. And then they go to the clinical laboratory where they have specialized tests that are really not available everywhere. So we have in England a few specialized referral centers. So they have transmission electron microscopy to cut across the cilia and look for structural defects, and then increasingly the use of immunofluorescent techniques to look for absent proteins. As the genetics expands and we understand what the motile cilia proteins are, then there's either a diagnostic point then, or there's an unclear diagnosis. So these are not easy doing. Even electron microscopy, we know that about 20% of patients just don't have an obvious structural defect, which is generally considered the basis of PCD. So actually, it— there's very few funded clinical tests for PCD, despite its importance. In respiratory clinics, so actually we don't do genetic testing at these earlier stages. And in England and in most places around the world, you're looking for a very well clinically defined patient and then subject them to genetics. And then if you don't find anything through the clinical tests and through the genetics, and they're still a bit mysterious whether they have PCD or not, then there's gene discovery research. And this is the team in London who's been involved in our celiopathy genetics. And at Great Ormond Street Hospital, the hospital that I'm associated with, they're currently providing a 122-gene panel based on Agilent SureSelect technology. So we have— GASS8 was a brand new PCD gene most recently cloned, and that isn't on the panel yet. But it will be in the next iteration. So we have all the PCD genes, isolated heterotaxy genes, and actually, because we can, throwing in the primary ciliopathies because of the ciliopathy interest at Great Ormond Street. Then in the lab, we have the 32— we have a similar panel where we've selected 651 genes, the 32 published genes, and we have various candidates from ongoing next-generation sequencing. Sequencing in human patients that we've done, or from genetic colleagues in Europe and abroad, the isolated heterotaxy genes. And we also put on 532 candidate genes, because this is a research panel, so from studies of ciliate model systems. So, for example, Cecilia Lowe has been doing an incredible job looking at the specificity identification of heterotaxy and situs inversus and cilia, motile cilia in the mouse. So we've got mouse genes, and Sudiptu Roy has done similar sort of global study looking at cilia motility in the fish, and we put some of those together to have candidate genes. So the ultrastructural defects that I've been talking about that underlie PCD, this is the cross-section of the cilia with the inner and the outer dining arms and the central apparatus and the radial spokes. And although we know that it has quite significant heterogeneity, there's only about 6 or so structural classifications for PCD at the moment, and that's just because of the level of resolution that you can get with a conventional clinical EM. So, the most common, and this is just statistics from the Brompton Hospital in London, 40% have an outer dynein arm absent, about 20% have both the inner and outer absent. There are various sort of microtubular disorganized features, either with or without the inner dynein arm. You can have the central pair absent, but quite significant numbers of patients have a completely normal cilium, but the beating is deficient. And then this very interesting new category, about 2% 20% of patients seem to have very few cilia on their cells. And this is called RGMC or oligocilia, just a reduced number, maybe 1 to 2 per cell. And so just to explain the genetics underlying those 5 categories of PCD patient, I've put the structures that are involved. So we know that the dynein arms get preassembled before they're imported into the cilium. They're assembled by quite a— well, an extremely complicated mechanism within the cytoplasm. And they are brought up into the axoneme by intravagellar transport. So outer dynein are so-called outer dynein arm patients. This is actually— that's the outer dynein arm. And all the structures in the cilium are multisubunit. So there's at least 250 proteins that make up the cilium. And this is an outer dynein arm with— of a docking complex and lots of dimeric components, light chains, intermediate chains, heavy chains. And these— what I've done is shown here all the genes that are implicated in PCD. And most of the time, if you lose one of these components, then the whole arm complex is gone. And then sometimes with the docking complex, or the that it's thought to be— the CCDC103 is thought to be to do with microtubular attachment. You can lose both arms. And then one very interesting protein, DNH11, is actually associated with no outer dynein arm, but a normal EM. There's about 10 genes, as I was saying, that sit in the cytoplasm and assemble the dynein arms together, and those are implicated in patients who have both arms lost, so why both arms are assembled with the same proteins, that is not known. There's one gene so far associated with the loss of central pairs, which is called hidin, and radial spokes, the radial spoke structures, it turns out that when you lose your radial spoke, you have an instability of the central pair. So, With these three genes, you can have a transposition where the central pair is completely gone and one of the peripheral dyneins shifts into the middle, so you get an 8+1 structure in these patients. And then there's one gene that encodes the radial spoke stalk, and if you have a problem with that— these are all head, radial spoke head, and they purely cause a central pair disruption. If you have a problem with the stalk, then you have a loss of loss of the central pair, and also a microtubular disorganization because loss of these radial spokes— it's like a bike wheel with the spokes gone. There's a huge disarray of the microtubules. And then what's most recently emerged, in particular with this new GAS8 gene, are a number of genes that encode this little nexin-link structure, which is also called the dynein regulatory complex. And when you lose those nexin-links, again, you can be microtubularly disorganized because the structure's gone, and lose the inner arm because the nexin is involved in inner arm attachment, or have something that's so subtle you don't see a defect at all. And there's 32 genes, so the other 3 are 2— one is the RPGR mutation that I mentioned causes the only known, clear-cut known syndromic form of PCSK9 where patients not only have a respiratory disease, but they're going to go blind in early life. And this is a little bit— the structural defects, there's only a handful of cases that have been reported, and actually most of them have no apparent structural defects in their respiratory cilia, and some microtubular disorganization has been reported. And then this new classification of motile disease has been termed reduced generation of multiple motile cilia, or RGMC. That's very long, so it's also called oligociliogenesis or oligocilia disease. And that's just caused so far by two gene defects, MCIDAS and CCNO. And you literally see when you fluorescently stain this, just one or two remnant cilia. There are a number of additional genes. I won't go into them in any level of detail, but they have been reported, but generally speaking, it's been a single family with no phenotype details or a question mark on whether there's respiratory involvement or just sperm involvement. Just a note on OFD1, there is a syndrome, and OFD1 can sometimes be reported as a PCD gene, but it's really a very distinct syndrome, Simpson-Golabi-Bemmel syndrome type 2, where there are OFD1 mutations that cause— they do cause a respiratory disorder with static and dysmotile cilia, but it's not connected yet with any sort of structural defect, so we believe that's a distinct disorder with macrocephaly and intellectual delay. So, when trying to understand PCD genes, which has really been booming in the last 10 to 20 years, we have great tools. We use all the usual tools that everyone does for their gene detection and isolating novel genes, but there is something special with the cilia that it really is specified by a limited number of proteins, and there's various cilia-specific resources where if you find mutations in a patient in a gene that you haven't heard of being implicated before, then there's lots of tools to be able to understand whether that is implicated with cilia motility in ciliate organisms, in particular Chlamydomonas, the very ancient photosynthesizing biflagellar organism. It's 3.5 billion years separate from us, but the flagella are hugely conserved. So all cilia proteins would be expected to be highly conserved. And that's very useful when you're trying to understand novel mutations and whether they're pathogenic or not. So we do our variant classification in a very standard way, and PCD tends to have quite clear-cut mutations that are, in a case— so with the primary ciliopathies, very often you have missense and hypomorphic alleles, and it seems to be the opposite in PCD. They're quite loss-of-function type alleles. So our genetic pipeline is to use that targeted panel. And if you get a biallelic disease-causing pathogenic sequence variants, then you can Sanger confirm and segregate. We do have biallelic sequence variants in a number of candidate genes, which we'll move into functional studies. And then with heterozygotes, or people who are unsolved using the gene panel, we now have the Genomics England project, and these projects are going to expand in number. There's one in America, and no doubt other European countries of course, so that's— that will be the pipeline going forward, because PCD is quite a major phenotype for the Genomics England project. And then to note that copy number variants do contribute quite significantly to the motile ciliopathies, and this is an example of a common Caucasian mutation in a DYX1C1 gene that causes inner and outer dynein arm defects. So we do a separate copy number variant screen, and we've used ExomeDepth because this was developed by Vincent Plagnol at UCL, and not to forget to look for copy number variants. The HYDIN gene I mentioned, just one gene involved with central pair defects, so when you're doing PCD genetics, the HYDIN does cause a problem. There's the HYDIN gene, which is on chromosome 16, and a virtually identical HYDIN2 copy gene on chromosome 1. Has nothing to do with PCD. It's to do with other phenotypes, but it causes literally a background problem. So it's virtually impossible by Sanger sequencing, we have found, any way to detect HYDIN mutations. And I don't know if it projects very well, but here's a control person with a GG homozygote, and the affected children in this PCD family are TT. But because of amplification of HYDIN2 at the same time, then they're looking like heterozygotes like the carrier parent. And actually, next-generation sequencing is about the only method that can distinguish the— there's enough variants to be able to distinguish the chromosome 1 from the chromosome 16 copy, and they align correctly, and you can detect mutations. But when you start to do PCD genetics, even in our gene panel, we see at least 50 to 60 60 SNPs and variants in HYDIN, they keep coming up, and some of them look like stop codons, splice mutants, so you can really get confused, and these are false positives due to alignment issues that you really have to hammer out by Sanger sequencing or other methods, and to keep a catalog of these repetitive, annoying HYDIN variants. And RPGR, as I've said, RPGR was reported as a mutation causing PCD some years ago, but it was always rather controversial, and literally only 2 to 3 to 4 more cases have been reported as to whether this very common cause of retinitis pigmentosa could also cause a problem in the lung. And for people in PCD clinics, the patients are coming to talk about their PCD, and they're not really expecting to be told that they're going to be going blind. We've got a couple in clinic, and it is difficult, and there are implications. So for PCD patients, because we're very early in therapy, actually the genetics of their disease, I would say it's not such a burning issue. We don't get requests for prenatal genetics quite so much for PCD, and people tend to live with their disease, but of course, if you're talking about an X-linked disease, then there's much bigger implications for being a carrier female. Half of your boys would be affected, and none of your girls. So, it doesn't affect the overall incidence, but it's something to think about in the clinic. So, I'm going to just briefly talk about our genetic survey, and we had 412 patients with DNA samples, 403 that undergone genetic testing, and 356 with completed genetic testing, and they very largely come from the referral centers of our national PCD service. So, it's a very nicely well-funded service for management and care of the patients since 2006, hugely efficient, but no genetic tests are funded within this service, and it's the same in most European countries. The genetics isn't considered significantly important, I think, until we develop more genetic therapies. So the distribution within the solved cases that we had does seem to echo the general distribution, with most of them having outer dynein arm defects, and quite a lot of normal, and the other microtubular defects being less frequent, and the loss of cilia being quite a low frequency. And so we found in 42% homozygous mutations in known genes, and in 26% compound heterozygous mutations in known genes, so 68% of success. And then unsolved we considered to be the heterozygotes or no mutations found at all. And we have a proportion with mutations in interesting candidates from our motile ciliome research panel. So 68% diagnosis is pretty high. In fact, it sounds very similar to what Enzo Maria was talking about for Joubert. And the 28— sorry, these cases of unsolved, there's certainly room for discovery of more motile ciliopathy genes. We found that gene panels have tended to be much more successful more successful than the various types of exome sequencing that we've done over the years. And the genetic spread is interesting. So DNAH5, that encodes a heavy chain of the outer dynein arm. This causes the vast majority of PCD disease, about 35% of disease. But importantly, if you stratify to outer dynein arm defect patients, then it causes 60% of that category of disease. So it doesn't cause any of patients with the central pair defect. So it's quite important with these stats to think about our genetic stratification. And I've just starred a few of the more common genes. They don't look very common, but HYDIN causes almost all of central pair defect, and these two interesting proteins involved in the molecular ruler of the cilium. They cause the vast majority of a microtubular plus inner arm loss defect. So the stratification is really coming on in leaps and bounds. I mentioned that PCD is really a loss-of-function disease, and that's definitely reflected in the fact that we get 30% of alleles— we have 512 alleles in total— 30% are non- onsense mutations, 32% frameshift, and 25% these somewhat more difficult missense and consensus splice site mutations. So, actually, we haven't really yet delved into the regulatory sequences for PCD, but it does seem to be primarily a loss of function of the open reading frame. So, just to summarize that, and I just have a few more slides afterwards, so we get quite a high success with a targeted gene panel. That certainly goes up when we look specifically for copy number variants. We found that the best gene panel, maybe a lot of people in the room agree, is the Agilent SureSelect, the most expensive tool. We have some common disease genes, DNH5 I mentioned, CCDC39 and 40, DNH11, and they are really causing a majority of specific classifications of defects, and a huge amount of loss of function. And interestingly, 42% of patients have homozygous mutations. Now, we have certainly got a prevalence of consanguineous families who have PCD, but it's not always the case, and we had a 2013 paper that actually a remarkable number of outbred, just Caucasian UK families, completely unrelated, have homozygous mutations in the same gene, and we don't understand that, why the mother and father are having the same mutation. It's very odd, and perhaps indicates more frequent— less about founder effects, but there may be regions of these genes that are particularly prone to mutation. I mean, I think we're a bit mystified by that at the moment. I haven't done a huge mutation list, but I will just have a few slides, to talk about some of the more common mutations and some of the things that they've revealed for the further stratification of motile ciliopathy diseases. So that the 3.5 kb deletion I mentioned in DYX1C1 is common in Caucasians, and there's a couple of more mutations that I'll talk about just now. So along with the huge impact of human genetics in PCD and in the structural world, Electron tomography is having a massive impact, and really new structures are being found in cilia because of this incredible resolution that you can get for the structure these days. And it's been much more in things like Chlamydomonas, but it is impacting in the clinic. It's far from being any sort of a clinical test, but this picture shows Amelia Schumach from the Brompton Clinic, and she's more or less pioneering tomography for patients where to try and tackle these very tricky 20% of patients with a normal EM. And sometimes they can have a quite normal-looking motility pattern, and they are just called difficult cases. So here's a couple. And these are nexin-link mutations. So this is the cross-section of the cilia. This is two of these peripheral dyneins here and here. If you can see the outer arm, the inner arm, and the radial spoke poking into the middle with a normal nexin link in a healthy control, and then the nexin link is destroyed by mutations in a couple of CCD genes, and that you have to do genetics or you have to do this incredibly time-consuming tomography. This took months to generate these pictures, so, you know, it's tough work. And then the— we are starting to understand a bit more about these sub-electron microscopy defects and finding that they do actually have structural changes, and I've just shown a couple of examples here. So the HYDIN, this tricky copy gene case— sorry. Yes, so the HYDIN was discovered to be just one little component of the central pair. The C2B is gone, and then the— I seem to have deleted my DNH11. So DNH11 was always associated with a normal structure, but we know that it's a massive protein, and it's a component of the outer dynein arm, and I can't show you it because I seem to have deleted it, but if you look by tomography, you can see that it's just the way that it's assembled into the outer arm makes it very difficult to see in normal cross-sections, and tomography does reveal those changes. I mentioned a common mutation in CCDC103, and this was quite a puzzling mutation for a while, and associated with a hypomorphic effect in patients. So CCDC103 is an assembly factor. It assembles the dynein arms, or it may be that the microtubule attachment— the exact function of CCDC has probably changed in the last couple of years to being part of the actual axoneme assembly. And a control person's nasal sample shows the normal beating, and people with a particular mutation, which was— and missense chains that seem to come up in a few families, they have a pretty much normal-looking movement of the cilia. And also, when you look in their cross-sections— this is a normal person with the outer and inner in a clinical setting with the electron microscopy— sometimes you see the arms absent, and then sometimes you would see retained arms. And then when you look at the conservation of that particular histidine at position 154, it's reasonably conserved in mammals, but it's, you know, it's a different amino acid in zebrafish, for example, which is a well-known PCD model. So we wanted to look at this more closely. There was a CCDC103 zebrafish mutant called the small hands mutant that came from the Tubingen screen. So this was worked on by Ian Drummond's group, and it has a classical ciliopathy phenotype with a bent axis cystic kidneys, and we could show that reintroducing the CCDC103 RNA fully rescued this phenotype. So looking at body axis curvature and also the movement of the cilia in the zebrafish nose, you can put a video camera up their nose and look at the movement. We looked at these two phenotypes and tried to model a few mutations, so the HISS154 4 pro is called M1 here, and then we had another mutation that turned out to be a polymorphism in these tests, and then a frameshift mutation. And if you look just at the M1, unlike the wild type, when you put the wild type gene back into the fish, it doesn't fully rescue either the body axis curvature or the movement of the nasal cilia. So it does seem to be doing something. And just to sort of display how this causes a problem in the clinic, So these are all patients with that particular mutation, and it turns out to be prevalent in the UK Pakistani population. So we now have about 15 families, and they, first of all, can have a really quite normal-looking nitric oxide. That very first screen that is done in the clinic can look quite normal. The EM can look quite partial, quite normal, and they don't always have cystinversus. In fact, we've found that there's quite a high level of cystinversus cytosol versus in patients with this mutation. We don't think that's because it's causing more cytosol versus. It's because they are the patients that are easier to pick up. So there could well be patients who are really not recognized with this mutation. I just talk about a couple of other things in my last 10 minutes. So we looked at a CCDC114 mutation in the Volendam population. Is an isolated population in Holland, and they all seem to share a splice donor mutation that creates an insertion where part of the intron between exon 7 and exon 8 is retained and causes a frameshift. And what we noticed was that unlike most PCD patients, in this very traditional isolated population, there's no infertility and plenty of parents with affected children. So, what was the reason for that? And what we found was that the CCDC114, which is a docking complex protein— so this is the outer dynein arm again with the docking complex. There's two homologues of the Chlamydomonas protein in humans. So, CCDC114 and human CCDC63 are very similar to each other. And when we looked at the expression, we could see that CCDC114 is very well expressed in the respiratory cells, in the respiratory cilia, and hardly at all in testis, in the sperm, whereas it's the opposite for CCDC63, very low in the respiratory system and high in sperm. And it does now seem, because a couple more genes are emerging with this sort of profile, where the sperm actually has a different composition. So this is slowly emerging that sperm may be actually quite different from our respiratory cilia in terms of their axoneme of the sperm flagella versus the cilium. And there may be some functional redundancy that can be important for counseling of patients if they're going to be told that they are not infertile. So just a note on this reduced generation of of motile cilia, and it really is interesting, and it's just in the last couple of years that this has emerged, and largely from the lab of Heimer Omran in Münster. He's done a huge amount for human genetics and really raising the profile of PCD. So this— the mutations that cause this syndrome, this motile ciliopathy, or loss of cilia, they have a very different function. They're not involved in cilia structure, but they're involved in the genesis of cilia. And this is a review showing the multiciliogenesis program, that cells exit the cell cycle to grow their cilia. You get an increase. So cilia are generated from the centrosomes. Centrosomal— the pericentriolar mass increases. You have amplification of the centrioles. They all migrate to the apical surface of the cell, and then they generate the cilia, and the cilia sprout out in these multiciliated cells. Mutations were found in human patients in multicilin and CCNO. When you knock down CCNO, this actually stops the— this is the xenopus model. So xenopus have motile cilia and mucus on their epithelial surface, and they're a good model for looking at motile cilia. So when you knock out CCno, you get many less centrioles, and when you overexpress multicilin, you get many more multicilia. But if you overexpress multicilin and you knock down CCno, then you can stop that multiciliation. So the conclusion was that CCno is working downstream from multicilin, and they all are involved in a huge cascade of gene expression that's that specifies the motile cilia. And so multicilin, or MCI-DUS, is expressed right at the beginning, and it affects the expression— it's a transcription factor, and it affects the expression of something called FOXJ1. It's a master regulator of ciliogenesis. FOXJ1 switches on the motile cilia proteins. CCNO is something to do with the centriola amplification step. So in patients, you get a reduced generation of cilia,, and in multicilin mutant patients, you also get structural defects, and they've lost their motor proteins, and the cilia are static. The 2 to 3 cilia per cell are static, whereas in CCNO, because it's about amplification of centrioles, then actually the cilia have a normal structure, but there's fewer of them. So there's a very subtle distinction. And so genetics has been very important, and it really isn't funded as part of the diagnostic diagnostic process at the moment, and it really should be. So, from what I've been saying, we know that the normal EM can be solved by genetics with mutations in certain genes, and certain genes can tell you about these difficult cases with hypomorphic mutations. You can actually target just two— you could Sanger sequence those two genes, MCI-DAAS and CCNO, if you have patients with oligocilia, or less cilia, and those are very likely— those are the major causes of that phenotype. And then the RPGR is still complicated, and only by genotyping and detecting an RPGR mutation are you going to be able to give the patient some counseling that they're likely to go blind as well. So we're getting some prognostic predictions. If you lose the central pair because of mutations in the genes that I've been speaking about, you don't have any situs abnormalities because central pair isn't present in the node cilia, and therefore situs is not affected. So many patients will not have situs inversus, but more importantly, they won't have congenital heart defects. There's lack of male infertility with the CCDC114 I mentioned from the Volendam group, but also, well, presumed that RPGR, because it's photoreceptor, won't affect fertility. Multiciliogenesis genes, I didn't really mention it, but there's a much worse lung function, much more hydrocephalus. They don't get size inversus problems because multiciliogenesis has nothing to do with the node cilia. They're not multiciliated. Inherited cells, and there's a couple of other indications that are published of genes that are associated with milder or worse disease for reasons we're not quite sure about. So, to wrap up, Afzalius, 30 years ago, had this hypothesis that Cartagena syndrome, PCD, motile cilia syndrome, all of it is directly or indirectly a consequence of cilia not being able to move, and perform their normal function, and he's right still. 30 years, we didn't say anything more than that. Even the ciliogenesis, they get their respiratory symptoms because of that reason. There's a huge stratification, and if you're interested like we are in this minutiae of the structural changes, there's a really becoming a stratification of these patients, and there's, of course, some gene discovery left, and we don't know how many Ps of PCD and oligocilia genes that will be in the end. I believe that PCD is a lot easier than primary ciliopathies. There are some overlaps, but it's really a distinct disease, and it's a loss-of-function disease, I think we can say. So the ciliopathies are massively expanded. We have certainly more than 100 genes and a really collectively a high incidence and a high medical impact for these diseases. PCD is pretty much of a common one, and whether you do or don't believe that PKD has implications for cilia function, that's really a major phenotype. So they're a really exciting field to be working in, I think, and that's reflected by the Cilia Meeting Series. So we have Cilia 2016 happening this year in Amsterdam. Amsterdam in October. Please sign up for that.

And just my acknowledgement. Thank you. So thank you very much for a very beautiful talk. Traditionally, there are no questions, but we can take one or two questions. There's two minutes left, two minutes left.

So for Elsa Maria or Anna, if there are any questions Yes, there is one question. Yes, hello, I'm Roxana Bachmann from Zurich. Thank you to both for excellent overviews. I had a comment/question for Enza Maria about the oligogenicity question in celiopathies. And so, with oligogenicity here I mean the fact or the idea that heterozygous mutations in two or more genes could actually cause a disease. And so with so many celiopathy genes now identified, and if you take Joubert, 30 genes, of course the likelihood that you find mutations or variants in more than one gene in one patient increases. And so I just wanted to share briefly our experience. So if— when we look at this, we actually find a small proportion, about 10% of individuals that have heterozygous variants in two or more Joubert genes, but if you look really hard, we're able to identify identify a second hit in one of the genes in a substantial number, or novel genes that were not, you know, one of the two. And the nephronothesis paper that you mentioned, actually, you know, by talking with Friedhelm Hildebrand, it turns out that almost all of those ended up to being solved by identification of other genes. And so it is my feeling at this point that there's little clinical relevance to oligogenicity in this sense, if it occurs at all. And so I wanted to know if you've looked at that and what your feeling is about this.

Yeah, thank you very much for this comment. I fully agree with you that the situation of two heterozygous mutations in two distinct genes, it's a very rare condition, it's a very rare scenario, okay? I think it is more going to be likely that we have like biallelic mutations in one gene, in a major gene, and then additional mutations may modify the phenotype. So this is probably something that we really need to look into, to have two distinct heterozygous mutations that complement each other and result in a phenotype. I agree that most of them, one of them is wrong and the other one is probably a second hidden mutation somewhere. Clearly, the reason, probably an excess of heterozygous mutations in ciliary genes, and we do need large numbers, and I think we do need to put together the cohorts and do some proper statistics and trying to correlate variants with phenotypes to see whether there is really a modifier effect more than oligogenic in the, you know, as we define it.

Yeah, I agree. Thank you very much indeed. There is a big need to put all the data together. Anyhow, it is 10 o'clock sharp, so we have to close this session.

And thank you to the speakers very much and for all of you to come this morning. Thank you so much.