Chapters Transcript Updates on Primary Ciliary Dyskinesia Diagnosis Amjad Horani, MD, presents on the diagnostic approach to Primary Ciliary Dyskinesia. everyone. We'll go ahead and get started. Thanks for joining Early Bird Ros this morning. Um, today, we have Doctor Orani, and he's specialized in, um, pediatric pulmonology. Before we get started, we just have a few announcements to please keep your cameras turned off and your mics muted during the session, and of course, there's always time for questions at the end where you're welcome to unmute. Um, as well, the QR code will be going up at the end for you all to scan for credit. We'll go ahead and give Doctor Ronnie our attention. Good morning, everyone. Um, my hope is that within the next 45 minutes, 150 minutes, uh, we'll review, um, general, uh, knowledge about this condition called primary cell dyskinesia, uh, as well as a bit of biology, uh, that kind of stands behind this condition, uh, specifically talking about motor cell biology. Um, so I'm the director of the PCD Center, uh, here at WashU. Uh, um, uh, assist in pediatrics, obviously, uh, but we do see patients up to The age of 23, 24, and we have colleagues on the other side at bars that um uh take over some of our older patients as they transition um care. Uh, our center also sees other conditions, not just the PCD we do see uh genetic lung conditions and uh uh what we call rare lung conditions. Um, anything that has a gene that drives it or anything that looks weird, we, um, love to see them in our clinic. Um, so the image that is shown in the background is actually a piece of human trachea, um, that was, uh, stained using a technique called immunofluorescent staining, um, showing the cian green, kind of decorating the, the top of, uh, the city. I don't know if you see my cursor here on the screen. But the greenies are these motorcia decorating the cells, and the cells themselves are stained with an antibody or a marker that stains the cytoplasm kind of shown here in bread, and this is a typical image or typical presentation of motor scia in the airway. So I do not have uh disclosures relevant uh to this talk. As I mentioned, uh, we're gonna spend some time talking about, um, uh, celia biology in general, then I'll switch gears and we'll talk about the condition primary cellulary dyskinesia, uh, a motor celeopathy, and towards the end, so we'll talk about some of the diagnostic tools we have for PCD and our approach for the diagnosis of primary uh cellary dyskinesia. So, uh, sta starting from the basics, uh, aera is an organelle um that protrudes from the top of a cell. Um, there are different types of scia in the body, but in large we can divide them into two main, um, um, types, um, ones that are motile, as shown on the left of the schematic here and ones that are immortile or non-motile. That we also call primary Syria, not to be confused with primary Syria dyskinesia. There are two different terms. Primary Syria are non-motor Syria and motor Syria are obviously are motor Syria. All of them share a common blueprint. These are the nine outer doublets that we learned about medical school. Um, um, but some of them had additional structures. Some of them are missing structures. If you look at the non-motor ya, they have the nine outer droplet are shown on the schematic. They're missing a structure in the middle called center pair, but they're also missing the motors that are important for motility. These are the extensions that look like crap arms that extend from the microtubules that are important for driving the motility of the cell. The motors, obviously, um, in their classical forms have the 9 + 2 arrangement, um, 9 outer doublet and 2 central pairs, and then the hoop, they do have the extensions on the microtubules. These are the motors. Um, the image, the scanning electron microscope on the far right of the screen shows an example of a primary cell extending from an airway cell. It's kind of small um protrusion out of the cell, and the scanning electron microscope on the far left shows that from motor cella they are typically multicellulated, so about 200 per cell, kind of look like a grass if you look at them from above. Now, primary era or non-motor ya, these are sensory organelles. They, uh, as I mentioned, they have 9 outer doublets as shown in the schematic. Um, they do not have the extensions from these outer doublets or the motors, and they're typically, typically 1er per cell. They're ubiquitous. They appear on almost every cell type in the body with the exception of uh blood cells and with the exception of dividing cells. Um, they're common in the kidneys, they're common in the bile duct, the pancreatic duct, in the cartilage, in the ears, in the eyes, and these are sensory organelles. They're like a cell antenna where the cell uses it to kind of uh sense what's around it, and it's very important during the embryo development. As I mentioned, the scanning lateral microscope, uh, on the right is actually of a primary area, uh, in an airway, and you can see how it extends from that cell. The small protrusions around it, these are called microbili, um, they're common in different cells and they have different functions. So in some sensory organelles or organs, these primary cia get specific sensory function. They, they are the um form the photoreceptors in the retina and the colorful image on the left shows uh cones, um um with the silicon protruding from the sensory uh layer of the retina, and they do sense light. Um, so if there's um uh genetic diseases that affect primary cell and the eyes, they do cause blindness. In the in the kidney, the primary cell are important for sensing um flow of urine. So when they are not working like they should, they cause diseases such as polycystic kidney diseases. And the ear, they form a structure called chinocelia, which is um as shown in the middle cell in that, in the cartoon or the schematic. Um, these cells sense a vibration and as uh obviously when they're defective, there's a bit of uh hearing loss as well as uh um a problem with Uh, vibration, uh, sensation. Because of the ubiquitous nature, um, genetic diseases that affect primary era cause syndromes called primary seropathies. Again, this is not PCD, this is not primary cyskinesia. This is a different, these are different diseases, and primary seropathies are multi-organ syndromes. Um, in their classical forms, these patients typically have a problem with their skeleton, as well as Problems with their kidneys and there is a lot, a lot of these primary sediopathies. Uh, the table on the right shows some of the more common ones, um, some of them, um, you've probably heard of. The polycystic kidney diseases are caused by changes or defects in primary cell in the kidneys, mutations in the PKHD one. both uh, um, uh, autosomal dominant as well as the autosomal recessive, uh, condition. Barne Wieder syndrome is another example of a primary seropathy. Um, there's a, um, a center here at WaU. Uh, these are patients that typically arrive, um, um, because of, uh, uh, um, uh, some developmental delay and, uh, some problems with their endocrine, uh, function. Um, and as other uh syndromes such as Rupert syndrome, um. But what's common in these syndromes are two things. One, many of them do involve the kidneys because of how important these primary cities are in the kidneys. Many of them do involve the skeleton, either with polydactyle, extra fingers, or even less fingers, and many of them are caused by changes in many, many genes as shown on the column on the right. These are not monogenetic conditions, but polygenetic conditions, but the beetle are caused by a group of genes mostly affecting the base of the cilia. Uh, Gert is caused by uh genes or proteins that, uh cause uh defects and structure called a basal body which is again sits at the base of the cilia. Many of these conditions, by the way, do not have uh defects in their airways, so they don't have um motor eropathies or um syndromes that cause PCD, although rarely they can. But for the most part, primary erliopathies are just sensory uh diseases that do not affect uh the airways. The second structure is a motor nodal cilia, and this is a very interesting structure. It's a single civia that appears on the cell, but unlike the sensory civia, these are motile. Um, so they do have the dining arms as shown in the schematic. They do look like the central pair and because of um the way they structure, their motility is um uh interesting. They actually rotate rather than move back and forth. And this structure or the uh no cilia appears during a very brief period during development around week 17 to 20 in humans as well as week 7 in mice, and they appear in a structure called the node. Um, the image on the far right, the top image shows a mouse embryo. Uh, with and the blue shows that structure called the node. This is a structure that appears for about 24 hours and then disappears. So if you're a researcher working with the node, you're unlucky because your structure of interest tends to disappear before you even plan your experiments, and in mice at least they appear in the middle of the night. But this structure is very important because it is, it determines. Body laterality. It tells the body which is it's right and left, and it does it in a very interesting way. The scanning image by the way on the far right shows a close up of this node, and you can see the um city kind of protruding in the middle of this uh uh pit or um um um. Depression in in the note. So as I mentioned, uh, it's an interesting structure. It has these uh seria that rotate, and this rotation it causes a movement of fluid in one direction, and the left wise movement of fluid as shown in the cartoon on the left, and this movement um triggers a cascade of signaling on one side of the developing body that tells that body, this is your left side versus the other side which turns to be the right side. So you can imagine that if these not the Syria do not move, they're static for whatever reason, then the body does not know where to put its right side and where to put its left side, and then we end up with conditions such as sits and versus totalis where the body just completely. Um, decides on laterality by chance. Um, oh, and in even severe cases, the body just gets really confused and don't put the organs at all in the right, um, uh, arrangement, and we get conditions such as heterotaxy. This is the reason why many patients with PCD, we'll talk about that in a bit, have satin versus totalis and why it happens in 50% of the patients with PCD with primary cell dyskinesia, because the body just decides at random which is its right side and which is its left side. The last type of era are the motile multicellulated cells, and from the cartoon as seen these are multiple sya, typically about 200 ya within the cell. They have all the typical structures, so the 9 + 2 arrangement as well as the dining arm or the extensions on the microtubules. Uh, these, uh, cells are movers, so they're important for either moving secretions, uh, uh, moving uh fluid, um, or propelling, uh, structures, uh, such as the uh tail of the sperm, which is a modified motor uh cilium. The video that is running in the background, these are from cultured airway cells show showing the rhythmic movement of the cilia on the cells. This and the uh scanning electron microscope is coming from uh cultured airway cells again showing cells that have multicilia. Um, around them are cells that don't have C. These are secretory airway cells. These multicellulated um structures are abundant in the brain. They're important for moving the CSF in the ventricles. They're abundant in the nasal uh sinuses, the. Tubes, so when they don't work, you get sinusitis and problems with the ear drainage. They're abundant in the airways. They're oviducts. They're important for moving the ova from the ovaries to the uterus, and they're abundant in the testes as well as the tails of the sperm. And when they don't work, we get a condition called primary pseudoskinesia, which is a motor celiopathy. So again, this is not primary seleopathy, this is motor celiopathy. Uh, the terms are a bit confusing, but they're two separate um conditions. This is a condition that we knew about, um, in medicine for many, many years, from the early 1900s. Um, early reports starting to come from Europe. Ebert was a Ukrainian physician that described patients with cysy verses, polyps and bronchitis, but it wasn't until the 30s when Cartagena collected a larger cohort of patients with cys, bronchiectasis and polyps that we as a community recognize this condition and thus the condition was called Cartagena syndrome for a long time. Then in the 70s, uh we um uh there was a link between the clinical condition and the uh defect in motorseria. Uh by Aylos, and in the 80s, the name changed from Cartagena into, to primary syskinesia. And this is the official name of this condition, Cartagena syndrome. I would not use it anymore to describe these patients. Uh one, because it doesn't describe all the patients. Um um as I mentioned, Cartagena by definition needs to have stats and verses. And not all patients with PCD have cytosine versus 50% of them do not. Uh, so Cortiner syndrome is not, um, currently is, is an old name and um should be avoided. Primary serviceskia is the official name. The images at the bottom shows, um, a CAT scan of a patient with PCD, an older individual with significant bronchiectasis. These are all these um um. Holes within the lungs. This is um significant uh bronchiectasis, and PCD can be really bad disease, um, not, um, probably as severe and sometimes even more severe, um, compared to cystic fibrosis in some cases. Uh, the chest X-ray on the right is a patient with sep and versus totalis, so the heart is sitting on the right side and the liver is on the left side. As I mentioned, PCD is caused by defects in the uh in the structure of motor cilia, and this is a snippet of the seminal paper, uh, in the New English Journal of Medicine by Eillis, where he described patients with the clinical condition, uh, um, PCD and um when he discovered that the structure of the era initially in the sperm, but later in the airway is defective. And it's the the images are not very clear, but if you look at the left, it shows a cross section of a normal city. There's like the 9 outer doublelet and the center pair, and the arrow shows the dining arm, the outer dining arm in this case. And in a patient on the right side, they have the 9 + 2, but there's no extension, so the missing. The outer and inner arm, which is a classical for PCT. PCD is a multi-organ condition. Um, it involves many, many organs and thus the symptoms, uh, reflect the, the involvement of these um uh uh organs. As I mentioned, they're still in the brain, uh, and then, um, if they do not work, we rarely get a hydrocephalus in PCD patients. This is typical of a few mutations that tend to be on the severe, um, um, end of the spectrum, um. Patients um have uh a lot of ear infections because of uh era in the ear. Um, most patients have uh significant uh ear drainage and significant, um, um, a recurrent otitis media at their age. Most of the patients do have sinus disease and, uh, as shown in the, um. CT scan on the upper right showing a patient with a thickening of uh uh uh the lining of the sinuses. Most of the patients, all of the patients actually do have uh a persistent nasal drainage. Most of the patients have a significant lung disease, uh, chronic cough, bronchitis, babies. Um, are born with respiratory stress in the newborn. About 80% of PCD patients actually present with newborn, uh, distress, uh, early on, uh, that, uh, can, can or cannot be recognized as part of the syndrome. Uh, patients, uh, male patients typically are infertile, uh, and female patients are subfertile because of the involvement of the era, uh, in the oviducts. And obviously, uh, patients can have uh situs abnormalities in their um simpler form. They have cys and versus totalis or flipping of the organs, but they can also have heterotaxy with significant uh congenital heart disease. So, uh, when we Get patients in the clinic uh with suspected PCD and suspected PCD can be because of the patient is presenting with this persistent cough that is not responding to say, asthma treatment. Or um recurrent otitis media at an early age or even a later age, an adult with recurrent otitis media kind of raises concern that there's something that is driving it, or chronic sinusitis that doesn't um respond to typical treatment, these patients come to the clinic and then we, we need to decide how to confirm the diagnosis. And there's a, a few uh diagnostic tools um at this our disposal. These are the current diagnostic tools that we use for PCD. Um, there's a, uh, an older tool called saccharine test that is not on the list, um, uh, and it's that's intentionally because it's a tool that is neither specific nor, um, uh, sensitive, and if, uh, at this point belongs just to the textbook. And we'll go one by one kind of an examples of these different tools, starting with high speed video microscopy, and when I say high speed video microscopy, I mean Taking images of Syria at about 200 to 400 frames per second to try to figure out if the city are moving correctly or not. This is a tool that in Europe at least is used as part of their diagnostic toolkit to confirm the. Diagnosis in North America, we're still lagging behind. It's only available in big research centers just because it requires specific expertise in recognizing the motility of the cia and in most cases does require culturing the airway cells in vitro or kind of outside the body, uh, to avoid, uh, non-specific changes. Uh, the video on the right that was moving a minute ago um shows a biopsy sample that I took from a patient with primary cyskinesia, and in this case the city are completely immortal, so that was an easy diagnosis because, um, you know, if they don't move, this is, this is likely PCD. Sometimes it's not as obvious. We have a lot of patients that might have only minute changes to the motility of the cia so it's not as easily recognizable. The second tool is the transmission electron microscopy, which has for a long period of time, um, been considered the gold standard for the diagnosis of PCD uh and it's obvious why PCD is a condition that affects the structure of a motorel, and this is again a reminder of the uh structure of PCD in the 9 + 2 arrangement, as well as the dining arm. So if any of those structures is missing, it's likely uh PCD. Um, and we do recognize different, um, um, changes. We can either miss the outer liner arm as shown on the far left, um, um, in some of the patients. We can miss both the outer and inner uh uh protrusions on the doublets or the outer and inner line arm. Sometimes the uh structure of the cross section is completely messed up where we have extra. Uh, um, tubers either in the middle or outside, um, what we call central pair defects, uh, and other conditions where it's completely messed up and, um, the structure starts to kind of migrate and move. Um. The TM is a great tool. The problem with TM, however, is that um if we do, if we send patients for electron microscopy and the electron microscopy result is completely normal, this, this does not rule out BCD, and the reason for that is about 30% of genetically confirmed patients with PCD have a completely normal ultrastructure just because it's not sensitive enough to detect some of the small changes. Um, Which is the problem if you're using that tool to rule in or rule out uh PCD. Um, the other problem with transmission electron microscopy is that it is, it requires significant expertise. Um, and the recommendation is to look at about 200 cross sections and, um, most of the time, there's not enough cross sections to look at or the pathologist, um, does not uh try to find this uh 200 cross-section because it's just time consuming. Um, so, Most of the centers have been backing away from using electron microscopy as their tool of choice, um, and it's only a few centers, um, that know how to do it correctly. Um, even here at WaSU, I have not been sending um electron microscopy, uh, routinely on most patients with uh PCD. The third tool is measurement of nasal nitric oxide. So nitric oxide is signaling molecules a gas that is produced by cells, and for some reasons the sinuses of these patients with PCD do not produce high levels of nitric oxide, and we've recognized. That many, many years ago and now we use that as a screening tool for these patients. Um, it's a non-invasive tool in patients that are above 5 years of age, we can do it. Um, all we need is just for the child to sit on the chair. We put a probe in the nose and we measure for about 20 seconds and we have a number. Uh, the graph that is shown in the background, um, shows measurements of uh different defects causing PCD and the levels of nitric oxide. You can see that uh the first three are different forms of defects of the dining arms, and all of them are low. There's now an agreed upon cutoff for screening of 78. The number itself is not, not that important, but below that number, it is likely uh PCD above it is becomes less likely. You can see that parents or healthy individuals um have higher numbers, typically the 200 to 300 range. Uh, it's an excellent screening tool. It has its issues though. One, some conditions, including cystic fibrosis, sometimes have low levels of nitric oxide. Again, for reasons that we do not understand. Uh, over the past 3 or 4 years, we started to recognize that patients with immune deficiency also have um low levels of uh nasal nitric oxide, which is a problem. Um, it's definitely a problem if you're using this as a screening tool because it appears does not differentiate between these conditions. And um we are starting also to recognize that some patients with um uh PCD have levels that are slightly above the cutoff that we've established. However, we're still using it as one of the tools because, you know, if a patient comes in with symptoms that are borderline and the nitric oxide is, say, 400, it makes PCD very, very unlikely. Now, the issue with not nitric oxide measurement is that uh this is not FDA approved um for many, many reasons, um, although it is actually recommended uh as a screening tool by the PCD Foundation. Um, so, um, it means that the, the machine itself is not available everywhere, uh, but, um, uh, it, I, I would assume, I would think that most uh big PCD center around the country. Have a PC, have a nitric oxide machine available and they can use it. We definitely have it here at what we actually do have two of these machines uh for diagnostic purposes. Um, 4th tool is immunofluorescent staining. This is very similar to the image I started my talk with, and it's a simple method. It's not very different than using immune force and staining to detect changes in cancer patients. So the tool is well established in pathology. It's way cheaper than doing electron microscopy. For instance, um, and, uh, in its simplest form, we just look for structures that's supposed to be in the city, um, put a marker and see if these structures are there, um, so we can see it or do not see it. This is an example, for instance, of a patient that have a change in the outer and in your arm. So we're using a marker called DNH5, which is a marker of the out arm. And if you compare the two images with the arrows, the patient is lacking this uh red halo at the top compared to a healthy individual. So this is a patient that does not have auto. Um, this is a tool that is, has not uh gained traction yet here in the US, but I think it, uh, within the next few years, it will, um, gain more traction, uh, simply because it's cheaper. Pathology departments like using immunofluorescent staining. It doesn't take much time. Um, and the antibodies themselves, um, can be acquired. There are a lot of commercial antibodies and they're not that expensive. Um, in Europe, um, at least in the UK it's, it's, it has integrated into their workflow for the diagnosis of PCD. And the last tool, which is the tool I like, it's genetics. Again, PCD is a genetic condition. So if you find change in a gene, you have the disease. Um, so it's, uh, uh, it can be uh uh uh in my opinion, it's probably the gold standard for the diagnosis of PCD. The problem again, nothing is perfect in this world. There are more than 50 different genes that cause PCD, and this is a list of many of them. Many of these genes are structural components within the er, other, um, proteins in the outer dying arm or proteins in the inner dying arms. Um, some of them are, um. Proteins within the cytoplasma are important in building the structure. Um, and, uh, because there are so many of them, um, sometimes it's, it's hard to, to use the right, uh, to genetic tool, and we'll go over the different tools available, um, uh, commercially. The ones I bought and read are um different genes that were discovered here at WashU uh cause of VCDOU1 is one of the earliest one that was discovered about 20 something years ago, but it wasn't until recently that we started to find patients uh with um mutations in FOA one that causes PCD. Um, This is just looking at the same uh genes but kind of arranging them with the ones that causes a clear structure defects. So when you send electron microscopy, you see a clear change within the cross section of the uh cilia and the ones on the right are ones that um The TM is kind of not neither here nor there. The change is just uh um uh minute, small, and can be easily missed. And you can see that's why uh it's becoming problematic to use electro microscopy for diagnosis because there's a lot of, uh, genes that not not necessarily cause a clear structural defect. Now, genetic tools, there are a lot of genetic tools. Uh, all of them are commercially available. Um, the simplest ones are using dedicated panels, the companies such as Evita and Blueprints that offer uh PCD panels or PCD panels in combination with uh uh congenital heart disease panels. These are excellent tools. They're, um, fairly sensitive. Uh, they tend to be on the expensive, uh, uh, side, uh, if not. By insurance, but they're problematic because they're a panel, and most of these panels have anywhere between 35 to 40 genes including included them, and I've already told you that there are more than 50 plus genes on my last count. There are 55 genes cause to BCD, so the panel. Um, uh, at the start, it does not cover every single gene that causes PCD, um, so it, it can miss some of the patients. The other problem with panels is that, um, depending on the way it is set up by the company, it might, um, miss parts of the gene. Um, this is, uh, they use probes to look at different uh segments of uh the gene, uh, they're interested in, and if the probes themselves do not cover the entire length of the gene. And there's a mutation that is not covered by the probe, that, that mutation will be, uh, will be missed. Um, ideally, uh, I think patients should just undergo um a whole genome or whole exon um sequencing. So this is kind of, uh, what is written on the right side. These are genetic tools that just looks at every single gene in the body and not um um uh restricted just to 30 or 35. So they're more comprehensive. Um, again, they're not perfect, uh, and they're not perfect because again, they're also based on, um, probes, and if there is um a, a place within the, the genome that is not well covered by the probes that were designed by the company, that also would be missed, but it is, uh, at least it covers most of the genes. Um. The other problem with with holding some or whole genome sequencing is that it generates a lot of data. So it um requires a robust um bioinformatics analysis uh on the back end just to make sure that we understand the the results. uh uh typically that, you know, the company should do that, uh, and my hope is that Uh, I hope that most of the companies do it the right way. Uh, but I think these tools are probably uh better use than just using a dedicated pen at this point and the pricing is uh sometimes even more competitive than um using dedicated pellets. Uh, when we send genetic, uh, tools and uh if you, uh, send a genetic tests, probably you've seen these, uh, um, terms used on the report. Uh, the report itself can come back saying, hey, we found two changes with, uh, in an autosomal recessive condition that are pathogenic, which means that we find changes in a gene that have been reported before associated with the condition you're interested in, and the changes themselves we predict to be disruptive, so they call it pathogenic. Sometimes they just call it likely pathogenic, which means that, um, you know, we haven't seen these variants before uh in other patients or they haven't been reported before, but the, the, the changes are probably very disruptive to the gene. This typically happens when there is um um a truncation of the, the gene or the protein in this case. So there is uh the sequence itself doesn't go through and instead of uh producing a full length protein, we're producing just a, a tiny. Uh, protein, and if you have tiny protein, it's most likely not working. So this is called likely pathogenic. On the other end of uh uh the conditions, these are benign and likely benign. It means that one, and, and benign is that we, we've seen these, these changes in a large cohort of health individuals that don't have the condition you're interested in. And the change itself in most cases is silent, that is, although there's a change, but the amino acid does not change. It's just the same amino acids. So these are benign conditions. Likely benign is, um, these are variants that also have been seen in a lot of uh individuals and when we run it through that, uh, uh, in silica or kind of uh mathematical tools, they predict that they're fine. The problem is in the middle, what we call uncertain significance or variance of unown significance, and I do a lot of genetics um in the clinic, and this is one of my biggest headaches is that you send, send, uh, you have a patient, looks like PCD for instance, you send for a genetic test and you get two variants of unknown significance. We have one of those patients that we've seen last week. Uh, variances of unknown significance means that um the tools cannot agree. Uh, either that variant, specific virus has not been seen before in patients. Um, it is not common in the general healthy population, but the mathematical tools cannot agree if this is, uh, disruptive of the protein or not, uh, which means that it is left to us to make that decision, which, you know, uh, it's not ideal. I would like, I'd like it to be black and white and not kind of gray and middle. Um, uh, and when that happens, we, we actually, the genetics is, um, not helpful and we need to rely on other tools and go maybe go back and do um TM or immune fluorescence staining or video microscopy to confirm the diagnosis. So, um, in the next few minutes, I'm just gonna show you maybe 2 or 3, cases of uh PCD, um, um, that are, uh, interesting. Um, so we'll start with, um, um, this patient. This is, these are, this patient, these are two patients that present, um, with variance in CCB 39 and 40, um. Uh, I'm lumping them together because they actually present the same. Um, um, in this case, uh, a 40 year old, uh, individuals right with chronic cough, um, uh, sinus disease, chronic sinusitis, ear infections was sitting on, um, the adult ICU actually waiting for lung transplantation because of significant bronchiectasis. Uh, he did not father any children, so he's also infertile, and we were asked to evaluate that patient. Um, um, we, in that case, we did both genetics and uh electron microscopy, and the electron microscopy shows, um, this unique, uh, distribution or disruption of cilia as shown on, on the, um, uh, on the left. So some of the cross sections shows us number one, look completely normal. Some of them look weird where there's extra ones in the middle, um 8 instead of 9. So this is called, uh, disorganization of uh Syria. Um, and, uh, uh, the genetic confirmed, uh, a mutation in this case, uh, actually in CCDC 40, um, these patients tend to be severe. They are as severe, probably more severe than cystic fibrosis. Many of them or some of them end up on the transplantation, uh, list, uh, just kind of to emphasize that PCD is not a benign disease. It can be really bad. Um, now, we are at what you are actually very interested in CCD 39 and 40 because of its unique organization. And this is kind of, um, and I promise you this is the only sciencey uh figure here uh on the talk. Um, so, um, we have a tool called single particle cryoM. Um, name is not important, but what it, what it means that I can um scan. Um, proteins many, many times and then actually using the microscope, see how the proteins look in real life. So what you see in the middle of this tube is actually one of these microtubules. So it's schematic on the far upper left. Um, shows the 9 + 2. if we take only one of those 9, so this is just what we call a micro, uh, doublet, and then lay it on the side and scan it many times, I can see, I can see it with a microscope. This is not um uh an artist. render of the microtubule. This is actually how the microtubule looks in real life, and the different proteins um are arranged in the way they're actually exists on the microtubule. Um, they're just color coded to make it easier to see. The CCP 39 and 40 is the golden or yellow ribbon in the middle. They're, um, they call on each other. That's why they're called CCD. They're called called domain. And they go across the length of the microtubule. And we, we found that in the past couple of years actually is that these, these um ribbons repeat themselves every so often across the, the microtubule and uh C uses them as zip codes or addresses for other proteins to sit where they need to sit. Which means that when they're missing, the, the cell doesn't know how to arrange its structure, the structure of the era, and then it gets disorganized. So that's, that's why the electron microscopy shows this disorganization because they, they lose the zip code uh addresses uh for um building the, the area. Um, other mutations of interest are in two genes called CCNO and incy again the names are probably not that important, but, uh, um, the phenotype is interesting because these patients, uh, do not have er at all. So, um, on the left is, um, um, a TM showing um motorce on the top. Uh, the patient just doesn't have any era and if you can, you can appreciate that probably better on the IF images, uh unfluorescent images on the right. Uh, number A shows, uh, a clump of cells with kind of the cilia on top of the cells and the patient, most of the cells don't have anything. Some of them only have one cilia. Um, these patients are very sick. Um, they also end up on the transplant list, um, and, uh, diagnosis is hard because, uh, you send electron microscopy and the pathologist say, I did not see enough um cilia to make a diagnosis. You send another one and said, Well, we don't see cilia and you know, for, for a period of time when we didn't recognize this mutation, we thought that we just didn't, we weren't getting a good enough biopsy sample and then we discovered that, you know, actually these patients don't have CT at all. The third one is just an example of a gene that is problematic called Heiden. We now think that this is actually the most common gene causes of PCD. However, it's the least diagnosed gene, and the discrepancy comes because none of the genetic tools out there look for hidden. The panda doesn't include hidden in it, and the holding some sequencing, the one that looks at all the genes. Uh, doesn't do a good job at, at fighting Hayden. And the reason for that is that, uh, during the evolution, the body decided that it needs another hiding gene. So there's two hiddens, Hayden 1 and hi and 2. He and 2 doesn't do anything, just sits there. It's a sotogene. So when you do genetic sequencing, um, um, the tool captures the, the, the pseudogene, so it's hard to see the mutation. Oh I'm gonna watch it. The other issue is that electron microscopy is completely new. So we're kind of stuck with, with these patients that we don't have a good way to diagnose them, uh, which is a problem. Uh, IF can can indirectly detect them. Again, as I mentioned, IF is not um um um widely available yet in the US. So PCD diagnosis. So you have a patient um comes to you, you suspect PCD. So what are the clinical criteria for the diagnosis PCD and probably this is um the most important uh slide in the whole talk. So, uh, these are shown here are the what I call the cardinal features of primary cell skin or the clinical criteria for PCD diagnosis. Um, and they're important. One, a patient, uh, a baby who was born term that, um, develop respiratory distress of the newborn. Typically they're born fine and after 4 or 5 hours, they start to kind of require oxygen and then they're stuck on oxygen for about a week time for no good reason. These are not uh um meconium aspiration or um premature babies. This is very unusual. This should raise the concern that this might be PCD. Obviously, on its own it's not enough, but it is one of the cardinal features. Any patient with uh lateral defect, whether it's heterotaxy or cytosine versus, this should really raise, raise the concern for PCD. Any patient that develops um chronic, almost daily cough at an early age, typically before they turn 1 year of age, many of them even before they turn 6 months of age, and they do not respond to the typical asthma management. This should raise concern for PCP. Any patient that have a lot of nasal congestion, again, that starts at an early age, that's throughout the year, winter and summer, doesn't appear to ever go away and, you know, it might vary day to day, but it's just always there. This raises concern for PCD. If any of, of your patients have all four of them, I would say 90% chance, maybe even a 100% chance that this is PCD. If they have 3 of these features, there's a 70% chance that they have PCD and if they have two of those features, it's about 50%. So these are very, very helpful for diagnosis, and we, we, we use them uh um uh every time in clinic. Uh, if we have a patient that has all of them, even if I don't find the gene, uh, it's, I call it suspected, highly suspected PCD. Now, uh, you probably noticed that ear infections, uh, did not, um, uh, find its way to the list and the reason for that is that, um, ear infections are common in pediatric population and so when, when, uh, a large study looked at features, um, um, it didn't, uh, reach the cutoff for um diagnostic differentiation. But again, uh, I would say most patients with PCD have a lot of ear infections. Most of them will end up with uh tubes uh before they turn 2 years of age. So these are the 4 coinant features of PCP. Unexplained just for the stress on newborn. That's the defects, daily year round but cough. Day to year around nasal congestion. Now, this is uh a bit of a complicated and convoluted diagnostic uh algorithm. Um, don't try to read everything, mostly because it's gonna change, uh, probably next year. This is the American Thoracic Society uh consensus statement on the diagnosis of PCD and it's gonna change because now both the American Thoracic and the European Respir Societies are working together to draft a new um Um, diagnostic criteria that hopefully should find the light within the next year or so. But, um, uh, using the American Thoracic criteria, you can see that it starts with the clinical features, the four cardinal features. If none of them exist, this is Not PCD. If one of 2 or 3 of them exists, then it's worthwhile, uh, pursuing diagnosis. In a center that has nasal nitric oxide machine, um, this is the first tool we use. Uh, we try to measure NO and then decide if it's worthwhile pursuing. If this is a center that doesn't have uh NO capability, you can see that we jump right away to genetics, not even electron microscopy. Um, electron microscopy is kind of somewhere at the bottom of the, the graph here, uh, just because of the problem of missing many of these patients. Um, if there is uh inability, obviously, we, we look at them, we look at the results, and then we make a decision whether we need to pursue other diagnostic tests, but most of the time we end up doing genetics. So genetics is becoming the tool for the diagnosis of PCD whether we do a nasoittric oxide or do not do nasoattric oxide. We find the patients with PCD, then what? What, what can we do to them? And we, we have issues here. We don't have um yet specific treatments for PCD but we have some treatments. Most of the patients end up needing every clearance on a daily basis. Uh, whether using handheld devices or manual, uh, chest physiotherapy or vest treatments, most of the patients require, um, a lot of antibiotic courses. Uh, every time they have an infection or the cough, um, changes and it doesn't resolve. Open its own within a few days, they end up uh on a course of antibiotics. Some of the patients are on inhaled antibiotics, especially if they have are colonized with pseudomonas. Nasal washes, uh, and nasal treatments, uh, especially if there are polyps is, uh, an important part of their management. Uh, as I mentioned, a lot of patients have ear tubes, even adults, um, adult patients will require fertility treatments, as I mentioned, most of the male patients are infertile and the females are subfertile. If the patients have a congenital heart disease as part of the PCD syndrome, then they um require a close-up follow-up with um cardiology, sometimes even heart transplantation. Patients that develop bronchiectasis end up uh on immune modulatory treatments with azithromycin as part of their treatment. Azithromycin actually has been shown to improve the outcomes of these patients on a large uh multi-center trial a few years ago. Um, some patients are sick all the time and we end up putting them on, um, um, preventive antibiotics. This is case by case, obviously. Uh, in her hyper uh osmo agents like hypertonic, um, it, it, it, it, it's role is debated. Um, I, I, most of my patients do not use this, uh, treatment. I, I think of all the patients we see, probably one or two use it. Um, um, it hasn't, uh, been proven to, uh, have an effect. Some of the patients are on pulmmozy, kind of borrowed from cystic fibrosis. Again, um, the, its effect is debated. It actually can be harmful in some patients, so it needs to be decided on a case by case, uh, uh manner. Uh, bronchodilators can have mixed results, so some patients benefit from it and some of them, uh, do not. And lastly, uh, although it's uh not available yet, uh, we are in the era of uh genetic uh treatment. Um, uh, there are, um, um, it, it's, uh, multiple. Uh, phase one and phase studies looking at gene replacement therapy for PCD, uh, with, um, I don't know the results yet, but my assumption is that within the next few years, we will start seeing uh gene therapy for this condition. Again, this is a genetic condition. So, uh, to sum things up, um, motoril are common in many organs. Motoril and the airways are important for mucocellular clearance, and when they are defective, we have this condition called primary cell dyskinesia. Um, not to be confused with the primary celiopathies, which are caused by the sensory cilia. Um, PCD is, uh, mostly diagnosed using clinical criteria, and we have some, uh, lab tools to confirm the diagnosis with genetic testing being, uh, the de facto gold standard now for the diagnosis of uh primary cell dyskinesia. So, uh, this is my last slide and the, the cells you see kind of swimming in the background, these are my cells. The uh scrape my nose and put them on the slide and kind of was, uh, uh, you know, they look nice and wiggle there. Um, our information for the center are showing the right, um, um, phone numbers for the, uh, coordinator Leslie, as well as the, um, Jen who, uh, um, um, help us bring patients in the clinic. Um, uh, and the website shown on the back is uh our website PCD rare lung, um, so, uh, some information, um, for physicians and patients as well as our contact information. Uh, happy to take questions. Well, thank you so much for speaking for us. Um, please, if anyone has any questions, uh, you're welcome to unmute or we'll give you some time to type it out in the chat. Also, I will pull up the QR code while we wait. Well, it seems that it was super clear, so that makes me happy. Um, thank you everyone for um uh logging in this early in the morning, and again, um, if you have questions, you have uh patients with PCD, uh, that you suspect PCD please reach out uh to me or um uh or one of, one of the uh other members, um, in the uh PCD center. We're more than happy to see your patients. Thank you so much for um speaking for us this morning. We really appreciate it. Thank you. Have a good weekend. You too. Bye, guys. Created by Presenters Amjad Horani, MD Assistant Professor, Pediatrics Division of Pediatric Allergy, Immunology and Pulmonary Medicine View full profile
