 I'd love to see if you saw that, and it would be really cool. So we'll get started. So I want to thank everyone for being here this morning, and most importantly, I want to thank Dr. Ran Allingham for making the trip from Duke to come up and speak to us this morning. Two housekeeping issues. So for the residents, if you can please contact Lynn, I'd like to make appointments with everyone just to go over what you're going to be presenting for residents' day. So like half an hour would be fine, and Lynn can look at my schedule and we can make it work. And then the other thing too, for those that are interested, the entrepreneurial faculty scholars, which is headed up by Glenn Presswich, has a program on February the 11th, which is basically benched to bedside, and it allows you an overview of how to take an idea, a drug device, and move it from an academic situation in a lab to actually a product that could be commercialized. So for anyone who's interested, that conference is a full day conference. It's open to everyone in the university, and that's on 11th of February, a Tuesday. So with that, I want to introduce our guest speaker. So Dr. Ran Allingham is the Richard and Kit Barkhauser Professor, Chair in Ophthalmology. He received this title in 2009, and he's currently the Director of Glaucoma Service at Duke University. He's a clinician, scientist, and researcher, and really one of the leaders in the field, in the field of glaucoma, and in particular the genetics of glaucoma. For the last 20 years, he's been at Duke, and he's focused his career on the genetics. He's looked at open-angle glaucoma, POAG, he's looked at pseudo-expoliation, which he's going to speak about, and also primary congenital glaucoma. And he's literally traveled all around the world to collect samples and see these patients. His undergraduate training in medical school actually did not start in ophthalmology. In fact, he trained in family practice. He trained at the University of Cincinnati in Ohio, and then went on to Washington Hospital, which I learned is in Pennsylvania. He did two years, three years of family practice, delivering babies and C-sections, and realized that this wasn't for him, and he really wanted something more focused. So he went into the genetics of glaucoma, of ophthalmology. He started his training at Eastern Virginia Medical Center in Norfolk, where he did his residency and then went on to do some research. Was it Massachusetts, INE, or Boston for two years, and did his glaucoma fellowship? In terms of what he's accomplished over these last 20 years or 20-plus years, he's a co-I, co-PI, on a large NIH grant looking at novel genes for primary congenital glaucoma. He's an active collaborator in the Neighborhood Consortium, which is a large GWAS study looking at POAG with Janie Wigs up at Boston, and he's also investigating the role of cerebral splinal fluid as it plays a role in both open-angle glaucoma and normal tension glaucoma. And in particular, the reason he's here today, he's been very, very active in exploring the genetic and molecular role of block cell 1 in the pathogenesis of pseudo-exfoliative syndrome and glaucoma. He's assembled a large database, and again we've been in discussions with him on how we can use the UPDB and the database that we have here to actually collaborate and add to that depth of knowledge, and he also has large databases around the world, which he's going to speak with us today. In terms of where he's traveled, I asked him for some pictures that were not necessarily medical, and these are some of the pictures that he provided. So at this point, I'm going to turn it over to Rand, and he's going to introduce why he loves the mountains and he loves this area of the country. There you go. Thank you, Barbara, for that kind introduction. I think, can you hear me with a lavalier, can people hear me in the back all right? I'd like to thank Alan and Barbara for inviting me to speak today. I just came down with an odd symptom myself. I have a little positional vertigo, so if I move oddly or I collapse, it's okay. I'm just dizzy. It's not from drinking. I have witnesses, so all right. So let me see if I can move on. Oh yeah, so well, anyway, this is some other things. So I do love the mountains. I'm quite a fan. I come to, my wife and I actually just built a home in Jackson Hole, not too far from here. So I'm in love with this part of the world. So I guess these are some of the other pictures. I guess I'm not on my talk, is what I'm not. Okay, well, this is Nepal, which we were talking about a little bit. Alan's been there. This is in the north. Where is that? And so how do I get, just do this? Oh, I see what you're saying. You wanted me to talk about these things. So yeah, so this was a population in central Luzon, Philippines that we had the opportunity to perform a small survey. They're called the ETA. I was very intrigued with them. I don't know if many of you are fans of the National Geographic Geneographic Project, but it's basically looking at isolated populations around the world and trying to figure out where they all come from. So I was talking to my Filipino research coordinator, Cecile Santiago Terla. And she mentioned this group of people in central Philippines that were African in appearance. I said, well, I mean, what do you mean by that, Cecile? I said, well, they look like they are from central Africa. I said, really? And they said, yeah, and they live up in the mountains and they're very isolated. So I immediately figured out that this has to be one of the original migratory populations out of Africa. And in fact, worked with Spencer Wells to study this group and part of one of the National Geographic documentaries was presenting this group. They're remarkable in their stature. As you see, my wife is Anna's in the picture. She's relatively tall, but these folks were like half hour height. When we were examining them, Anna is a clinical psychologist. We gave her the job of dilating the patients. So she actually threw her back out by leaning over and putting the drops in the patients in their eyes when she was dilating them. I said, well, Anna, why don't you just ask them to stand on a chair? She says, I was. So they were remarkably wonderful people, very, very colorful, very, very friendly. I have to say, it was just a special, special group. OK, so now I'm done with that. But we actually were interesting. So we found that they had exfoliation syndrome in this population is about 6%. It was difficult telling what their ages were because they had no calendar. And so we used the reference of World War II when the Japanese had occupied the Philippines. So they were very aware of that date. And then we kind of went back to that early 1940s time point to figure out approximate ages. So some interesting problems along the way. So today I'll be focusing on exfoliation glaucoma and syndrome. It's something that I've been interested in for a very long time. I looked and did some family studies in Iceland in the late 1990s. And I've worked with a variety of really wonderful collaborators over the years. I have no financial interests or conflicts. I'd love to have one, if nothing other. One day maybe I will. But actually, in the sense that it would be great to have a treatment for this disease or others. And primarily in the talk, I'm going to have broken it up into four basic places. I'm going to start with the historical context of the disease, do the clinical pathology. And then I'll talk about the etiology and some of the genetics that we'll be talking about later. So it all started actually with this gentleman, John Lindbergh, in the early 20th century, who was a Finnish ophthalmology resident. So how many ophthalmology residents do we have here today? Wonderful, wonderful turnout. So I'm sure you'll bond with this experience. So he really had a very deep inclination to study Aksenfeld syndrome, which had been recently identified in that population. The problem was that he didn't have a really good way to look at their eyes. So actually, he did, I'm sure, what any of you would have done as a resident. He built his own slit lamp. And so he did that, and he had some pretty good help. He had Alvar golstrand. You'll remember the golstrand model eye. I guess that's still important, right? Well, it became a Nobel Laureate, one of the few in ophthalmology. But it was his design that he actually used. This is not his slit lamp, but it was something like that. So you can only imagine what it would have been like practicing ophthalmology without the ability to see what you were studying. His first of these are actually his drawings. So his first description was actually very good. You can see the typical bullseye pattern described the grayish flakes on the lens surface and other structures that were associated with it. He also noted that Glaucoma was present, at least in his group, in about half of those who had this syndrome. And that it occurred in folks that were older in age, usually over the age of 60. He published his work in 1917. And he shared his thesis and these ideas with Alfred Boat, who was, of course, well-known, probably one of the most famous ophthalmologists of the time, in Germany at a meeting. Dr. Boat proceeded to publish three papers about this syndrome. He did fail to mention our young Dr. Lindbergh. And it took literally 20 years later when one of his countrymen pointed out to the fact that, in fact, he had identified and described carefully the syndrome some six years before the first other published report. So something kind of a little historically interesting. So as far as clinically, I don't know any Gary Larson fans here, I always kind of like the obvious here. But the read, you say, why is this slide up here? So it is that with exfoliation syndrome, it's actually pretty simple to see if you look for it. And there's very few things that it can be other than exfoliation syndrome. So all right. So as far as the epidemiology, it's found virtually in every population in the world, the Alaskan Inuit. It was not identified in that population. But I wouldn't be surprised if they have a case or two up there. But India, the US, actually the Navajo population, we were talking about that last night, has a very high prevalence. And I think this is over age 50 or so of exfoliation, which is very interesting. In Africa, African-Americans virtually have none. And the only individuals of African ancestry that I've seen that have exfoliation in the country when they knew their ancestry were actually from Ethiopia or other portions of Africa, but not West Africa. I've done a lot of work in Ghana and worked with Don Buden, who did the Tema Eye Survey. Between the two of us, we've examined at least 5,500 people in Ghana, West Africa. And between us, I've never seen a case of exfoliation he saw two. So it's virtually nonexistent in that population. However, if you go to a different part of Africa, South Africa, it is actually quite common. And so 8% of those over the age of 40 have exfoliation in South Africa. And we're gonna get back to that later in the talk. So exfoliation syndrome, as far as what does it mean? It means very high risk for glaucoma, increases risk at least five to 10 fold. In the longer you wait, the higher the risk becomes. About 25% will ultimately develop elevated intraocular pressure, presumably from the incorporation of all these proteins in the trapezoid mesh work. About a third with ocular hypertension will go on to develop glaucoma. If you have a patient with, patients with syndrome versus patients without syndrome who are ocular hypertensors and you match them for age and for IOP level, actually the retinal nerve fiber layer is thinner than those who have syndrome than those who have the matched ocular hypertensive group. So one of our past fellows, Josh Stein, who is now at the University of Michigan is doing some really cool work looking at the Medicare database. Ken Smith is here, one of the big databases to look at data around the country was the first to really note that there appeared to be an effect of risk based on latitude. And he found in his population looking at ICD-9 codes and what have you, that the northern tier had about 114% increased risk of exfoliation and the southern tier about a 17% decreased risk. In a separate paper by Kang and Associates with Lupa Squally found that if you were in the middle or southern tiers of the United States, your risk of exfoliation syndrome was about 50 to 75% of the average. So the factors that increased risk of exfoliation, a low ambient temperature, sunnier weather, higher altitude, actually not Scandinavian ancestry, which was interesting. So does that sound familiar? So all of you guys get your eyes checked, right? As you're living in the ground zero exfoliation. Now, if you compare exfoliation, open angle glaucoma to primary open angle glaucoma, typically the IOP is higher, there's more advanced optic nerve damage, and there's worse visual field loss in that group. You also, the optic nerve damage and the visual field loss also progresses more rapidly. And it responds somewhat less poorly to medications requiring surgical intervention more commonly. Laser trabeculoplasty, at least in my experience, seems to be actually a little more effective in this group. I don't know, Alan or others, you kind of agree with that. So whether that has some interaction with the material that's present in the trabecular mesh work or not. Obviously, I think many in the room are familiar with the risk for cataract surgery. It raises that risk quite a lot. The pupils dilate poorly, the zonules are affected, are unstable, the lenses may sublux, and risk of vitreous loss is somewhat around 10-fold greater in those who have syndrome, births and the nose who don't. So approaching them differently is wise. So what we see pathologically, here's a very typical appearance of the nerve. You have this central disc and then an area where the iris has rubbed off that protein on the surface and then a collection of protein here. Sometimes this will scroll off. The zonules, this is a gonioscopic view and this is a pretty typical appearance if the sensation's dilated, if you use your goniolens, you can look just over the lens. You can see they're clumped, they're some that are missing. So this is at the heart of the problems involved with cataract surgery as well as the capsule having a different, maybe it's a more fragile capsule as well. If you look at the histology, you can see this is a lens capsule. Whoops, pardon me. That's the first time my phone's ever gotten off when I'm giving a talk. And so you can see this material, it just seems to be coming off in plumes of the lens capsule. Looking at the transmission electron micrograph, these are kindly offered to Ursula Schlosser-Schreihard in Erlang in Germany. You can see that this is stained for the exfoliation material, actually, lox. And you can see that the lox protein is being exuded and deposited in this material outside of the cell itself. It's very interesting. You literally can look at the vesicles right here heading up to that site being beautiful, beautiful work. On the iris you have exfoliation deposits around the pupil. There's vascular changes within the iris stroma, and the pupil often has what we call a moth-y-tooth appearance. If you look behind the iris in a donor eye, you can see here the ciliary processes and they're just snowy white. So anyone doing endoscopic laser of this region, I don't know if anyone's tried that, it doesn't work very well. It's like a shield, it's white shield. It's just very hard to get any treatment whatsoever. You can see the zonules again are kind of fragmented here. There's also exude exfoliation material in the posterior surface of the iris. But none is seen beyond the auras serata posteriorly. There's increased pigmentation of the angle which we're all familiar with. This is called the sample E.C. line. And it actually can be mimicked by a number of conditions where you have excess release of pigment. There's also exude exfoliating material. We just saw that posteriorly, but you see it's coating. It's on the surface of the ciliary body and actually on the posterior surface of the iris. So there's a lot of that material in the central part of the eye. Looking at the optic nerve, the elastotic degeneration is the term that we often use. This is a control lamina crebrosa. You can see the collagen fibrils and then these little very discreet packets of elastin. Typical amorphous appearance, but you see this is the disorganized appearance of a lamina crebrosa in the patient with exfoliation glaucoma. You can imagine that that might have some major impact on those axons as they try to traverse through the optic nerve to the brain. And if you compare exfoliation with POAG, there's far more of this type of change in exfoliation than there is in primary open-angle glaucoma, the most common form. And then of course it extends well beyond the eye, so it was realized relatively early that it's the skin, multiple organs, brain, heart, lung. There are systemic conditions, which Barbara and Alan and Ken and I and a group are trying to explore if there's a way we can look at that with the tremendous database here at the Mariana Eye Center and at the University of Utah. There is some suggestion that vascular disease is more common as well as sensing neural hearing loss. So let's look at the etiology here. So we have environmental influences, so what actually causes exfoliation syndrome? Trauma initiated, some of the very earliest cases of exfoliation have occurred in people who've had corneal transplants or ocular trauma. Though the youngest cases I'm personally aware of in the early 20s or maybe late teens or in those patients, to my knowledge, all had some type of injury or transplant. UV exposure appears to be important. Dietary factors, Lou Pasquale and his group found that maybe coffee ingestion could be related to this. He noted that the Scandinavians drink something like six times more coffee than the rest of us do on average. And then infectious factors have always been suspected for the reasons I just described as far as penetrating care to plastic. And then there's geographic clustering that we're aware of and population clustering and then evidence of familial transmission. Well, on the road to figuring out what causes this was Iceland. Iceland is kind of a unique experiment. The population since the Vikings were running around has been pretty stable since 1200. So it's basically very few people move there and very few people move out. It has a centralized healthcare system and it's an excellent population for genetic studies. It sounds similar to what we have. You have here on a much grander scale in a different population. A company decode was a biopharmaceutical, very successful in its day, was devoted to gene discovery and co-opted the whole country into the grand experiment. So it was done with consent of the population, at least a popular vote. And I'm not sure this would ever happen in the United States but so they apparently did agree and the company agreed to share the profits and the health benefits with the Icelandic people that were to be discovered. They did in fact find many, many major discoveries in diabetes, cardiovascular disease, macular degeneration, a lot of smaller syndromes, but a kind of that sharing the profits thing went down the hill when they went bankrupt a few years ago. But what they also reported in 2007 was a genome-wide association study of exfoliation syndrome and glaucoma patients in the Scandinavian population. The initial data set was in Iceland and then there was a corroborative data set in Sweden and what they found was a single strong association with a 90% genetic contribution of this gene, lysol oxidase like one, LOX1, LOX-L1 is what we'll refer to it as. They found three single nucleotide polymorphisms or SNPs that were associated with that too were coding protein changes and one non-coding. And they also found, yep, there was really no association with primary open angle glaucoma which was of great interest at the time. So in fact, supporting what many of us thought, this is a separate disease, this is not like primary open angle. But what does a GWAS do? That's a genome-wide association study. Well, it provides a very strong location point for genetic variants, but it does nothing about telling you about a disease mechanism. And so translating this genetic information to what in fact has gone wrong is really where most of the work takes place. So what I'm gonna talk about is some of our progress and those of others in this journey and what those implications actually are. If you look, this is one of the rare diseases where you look at the gene that was found and you say, golly, this makes sense. The LOX-L1 is a key enzyme in elastic fiber formation and stabilization. It actually catalyzes the polymerization of tropo-alacin into elastin polymer. So it's a real key player in everything that we know about exfoliation syndrome. And then the question of course was, well, you have these coding variants that change proteins from a leucine to an arginine and asparagine to a glycine. Is that what's going on in making this normal appearance turn? To this, if you look at the LOX family proteins, the first to ever describe was LOX itself, Lysol oxidase. And it is the protein that is responsible for manufacturing all that elastin in our bodies. Elastin is a pretty cool protein. It has an amazing ability to snap back, hence the name. We didn't have elastin, we would just break or we'd be like little robots like this. The elastin is made only in the earliest part of your life. After the age of the first few years, we don't make any more elastin in development. And we keep that elastin for our lifetime. The half-life of elastin is about 70 years. These guys help fix what we have. So LOX-L1 does that. You can see if you just look at the structure that they share this common copper binding enzymatic motif which is used in all the synthesis of the propoelastin to elastin and other players. And then they have a lot of different signaling molecules that sends them to different places to do their job. Another interesting feature about LOX-L1 is if you look at elastin fibers, and this is a stain for elastin, you actually find that LOX-L1 not only catalyzes elastin, it has become a component of elastic tissue. So that's kind of unusual. You would think it would just build and move on. It actually gets taken in, and you can see from this merged photo just how closely that corresponds. Now if you look at trabecular meshwork tissue and you stain for a LOX-L1, you can see that LOX-L1 is this red and it's staining, this is a trabecular meshwork here, here's a ciliary muscle a little bit there. Here's Schlem's canal, it rings Schlem's canal, and here are some of the aqueous veins. So LOX-L1 is actually distributed all around that portion of the eye. So as I mentioned before, we had two coating snips that alter these two amino acids in these positions that are highly associated with exfoliation, and we had this one non-coating sniff, which means it is a change in the DNA, but it's in the intron, and so no, no, it's not translated in their protein, and it does not alter the protein structure. Well boy, there's a lot of interest when LOX-L1 was discovered. So everyone who had exfoliation patients' data sets wanted to check it out. And primarily because the other coating sniff, the non-coating was a pain in the butt to actually type, most people just typed the two protein altering snips and found that, wow, well this one, you have the amino acid that was the risk amino acid in the population was in the same as the Icelandic group, but then you looked at these populations and it was reversed with this particular variant, but this variant, they were all the same. So maybe that's the protein variant that causes a change in the protein that causes exfoliation. Well, we went to South Africa to see what was going on there to see if that was also true there, and we found out in fact this one, all of the outside of Africa populations had the one variant as a risk variant in Africa, it was reversed. So actually the other amino acid was the risk amino acid. You say, well what does all this stuff mean? All this means is those snips in the DNA of these patients, they're markers, they're near the action, they are not part of the action, so they are not, we call they are not functional, they are not coding for a functional change. So what it actually suggests is at the end of the day is that the protein lock cell one is fine, that's not the problem, it's regulating that protein that probably is the product. And then to hammer another nail in the coffin, actually looked at the two protein variants in both and looked at it in cell culture and they basically behave exactly the same. So there's no evidence to support it's a protein thing. And this supports the fact that maybe it's an expression problem. So if you look at the laminar kerbrosa tissue, you say, okay, I wanna look at what's expressed. Here's lock cell one, elastin, and the microfiberal system, all involved in elastic tissue. You can see that if you compare it to controls, there's much less of all of these things. So you have down-regulated compared to controls the entire elastic tissue generating architecture and mechanism. So this is the gene, we're not gonna go crazy on this, so hang in there with me. It's got seven exons and these are the exons. Here's exon one, there's a promoter for exon one and it's transcribed in this direction through here. Now that's, here's the location in this gene. This is the DNA going from here to here. This is the location of the first two coding SNPs. You can see they're located in the exon so they would change the protein that makes sense. Actually, the SNP that was most associated with exfoliation was the third one and it's located in the intron but very nearby. So we were kind of interested in that. We were also very interested in the South African population. They had tremendously powerful association for these SNPs in that population. So we went ahead and sequenced the entire gene. We sequenced all those exons, we sequenced all the introns, a great effort. It took a very hard driven medical student two and a half years to do that and we sequenced some of the promoter region and then we did an analysis of all the variants between cases and controls of those two groups. Those with exfoliation and those that did not have exfoliation. So in this particular image, you can see this again is the gene right here. That's exon one, two, three, four, five, so forth. And these are all, this is the association value. So it's done in a negative exponent but this is 10 to the minus 12 p-value. That's a pretty high p-value. And this is where we were here and keep in mind this was only 40 cases. I mean, actually it was 25 cases and 25 controls. So this is the intron one. This is the location of the original SNPs right here and the association. You see that the one that was the highest association in Iceland is now actually one of the lowest in the African population showing it is just a marker. But if you look at this region, this is really important. This is where all the association is located. It's saying X marks the spot. That's what you wanna know. So what we did find, we said, well, golly, what else goes on in that intron? Well, there's a whole group of RNAs called long non-coding. They're called LNC or link RNAs or NC RNAs that actually never become a protein but they are massively involved in making all those proteins work. And so it's actually in the opposite direction and some people find this rather amazing but DNA codes in both directions. In fact, you can have genes interspersed with each other and that's actually very common. I think in my naive thought years ago is that you just have genes and they're all lined up. Here's one, here's two, here's three, nothing like that. They're all over the place. So you have this one and one of the types is called an anti-sense RNA. And it's called an anti-sense because it incorporates some of the coding in the opposite direction to control that gene. They are regulatory. They typically, at an anti-sense, typically reduce the target protein's level. But it's the body, the body has a way of saying, yes, make some, stop making it, start making it. Without that, we'd be one big mess. It'd be a blob of protein on the floor. So then we decided, how do you test this region? Because the reason of the region of interest is actually this intron right here. So this is where the promoter is for the anti-sense. So we say, well maybe that has something to do with it. So you take that promoter and you take the associated region that we've talked about. We attach it to something called Luciferase and we do a reporter assay. You say, does this drive this process and you get a lot of high Luciferase activity? Very simple test. So you transfect cells and you do this. You do kind of Luciferase testing. So we took this region, that's that high association region and we simply broke it up into DNA fragments of varying length to see what does it do to this reaction. We attach those to Luciferase and this is what we found. This is just a vector, it's a control. So this is, one is how much Luciferase you make if you do absolutely nothing. And this is adding this segment then this segment and so forth and so on. And this is the entire length of that risk segment that we had identified earlier. So what you find out is if you use these segments, these pieces here that actually it does in fact act like a promoter. So it increases the amount of the Luciferase assay in this area. But what was very interesting is that when we included the entire risk segment, it completely eliminated the production, it completely shut the promoter down. And this is actually the risk part of the gene. So we're seeing what's going on. It looks like it eliminates the promoter of the antisense. So right there, so there we go. Complete risk and there's no promoter activity. All right, so what are the implications of all this? So the highest region of association includes the loxal one, a antisense promoter, we just said that, that the risk DNA sequence reduces or essentially shuts down that promoter activity. So reduces the antisense. So it actually implies that you shut down the breaking system for the loxal one gene. It actually suggests that you have too much loxal one around when you have exfoliation, which is somewhat counter to some of that recent data that we were looking at. But actually Ursula Schlotzer-Schrayhardt had looked at this before. Her very earliest examination of this, if you look at control, and this is just ciliary process expression, if you look at controls, you see that loxal one expression is here in exfoliation syndrome. In pseudo exfoliation, it's actually elevated compared to control. And then as you go on later in exfoliation process from just syndrome to glaucoma, that expression drops. So this actually agrees completely with what we found, that initially there's too much loxal one around. And then down. This is some of the immunohistology looking at loxal one. This is control, and green is the loxal one. This is early exfoliation in a ciliary process. You see just a little bit of the material right here, but there's a lot more staining here, and this is late, and you see that staining intracellularly is gone, but you have lots of it laying on the surface. So it may be in fact that as this accumulates and builds on top of the surface, that it shuts down that machinery. So what are the implications of this? So you have molecular data that suggests loxal one expression may be greater in exfoliation risk cases. The current data conflicts with that, but it may be that early in disease, this is a different story, which we also just showed. That exfoliation is clinically diagnosed with visible proteins, so nothing is known really of the preclinical exfoliation state, because we wait until we see the protein to make the diagnosis. So what do we do next? So this is kind of the concluding thoughts in where we're all going here. So identification of functional sequence, and we need to find what is in fact functional here. We have found a region that appears to have a functional role, and so we're now currently analyzing large population data sets in Japan and actually the original decode data set and actually vary about every continent, except Antarctica, we have DNA from in conjunction with Tinong in Singapore and the Icelandic group, and we wanna determine the molecular and cellular mechanism in cells and tissues and of course ultimately in the animal model. So the human studies, we could actually calculate the actual disease risk on family and pedigree studies if we know exactly the code that we need to look for, which we don't currently have. We can also explore the environmental triggers for this disease knowing who is in fact at risk and who is not. And we could also do that certainly that we can correct and we know for age, population, environment. We can do a lot of studies we can't currently do now. And we can look at the role of systemic diseases if we know what's functional as well because many of the people that we're studying are younger than the age that normally would demonstrate exfoliation syndrome at all. It seems to me at least and others that lock cell one and exfoliation glaucoma syndrome is the ideal disease to target for gene therapy. It's common, it's blinding, it has a major phenotype, it's readily diagnosed on exam. There's a dominant genetic target, which is lock cell one. Again, this gene is responsible for 90% of the genetic effect of the disease. We contrast that with primary open angle glaucoma and we're still counting. So we have five or six that we can see and we've replicated but there's many more than that coming down the pipe. Furthermore, some of you may be familiar with the genetics and diabetes. In diabetes, there's probably 150 genes now currently known that contribute to diabetes. Each one contributes just that much risk. So it's a mess. What would you try to genetically alter when you have 200 genes to deal with? We have primarily one. And on top of all that good news is the anterior chamber. The big phenotype is glaucoma in the lens. It's in the front of the eye. Well, how easy is that to get at? So it's highly accessible and the trabecular mesh work, which is a phagocytic organ is sitting right there and that's the key tissue that we really need to treat. So and then lensonials will throw that in possibly and it's not in the retina. So we have a perfect situation of a common disease, a common gene and a very easy way to get at it. So we can certainly think of viral vectors. This is an example of Pedro Gonzalez in our lab who's looked at a targeted adenovirus for the trabecular mesh work. This is a donor eye looking from the inside out and you can see the staining of just the trabecular mesh work with this viral vector. So put in what you want in this vector, inject it in the eye and the TM will say thank you and take it right up, pretty nice. We could do gene silencing. That's when you use special RNAs to shut the gene down or make it work better and conventional medical therapy because keep in mind again that lock cell one is an enzyme. It can be altered by conventional therapies or we may want to alter other gene proteins but we can use eye drops for this potentially as well which makes it so you're not dealing with the systemic risks of what we'd be trying to do here. So in conclusion, we know that XFG is common. It's a major cause of blindness. The biology resolves around lock cell one. Alteration of gene regulation is largely responsible and the key disease tissues are in fact readily accessible and amenable to genetic and conventional therapy. So I want to thank you at this point in time. I appreciate your attention. These are my collaborators all around the world. The list is growing and growing. And particularly at Duke, Mike Houser is a strong and Pratap Chala and Dan Stamer, Tin Ong in Singapore. Susan Williams was a wonderful early graduate just out of residency who did the work in South Africa. She then went on to get her PhD in genetics. She's just wonderful down in Johannesburg. Her chair there is Trevor Carmichael. I don't know, Alan, if you know Trevor. And then Robin Rautenbach is at another institution who also did another, contributed to this work and her own. Thank you very much for your time and attention. We can turn the lights on for those of us who can wake people up at this point. Ran, thank you very, very much. And it's always nice to hear a presentation on genetics that they can actually follow. So it always becomes so complicated. But we can open the floor to questions and Bala. As far as the gene itself in keratoconus, that's a very good question. So loxal one, again, is expressed all over the body. It seems to be a protein that is produced whenever there's trauma. And so, and Bala's question is, well, what about keratoconus and some of these other diseases? Loxal one, to my knowledge, is there's no association with keratoconus. But I would think it's highly likely, in fact, that it's involved in that process. But to date, we don't have, loxal one has not been associated with any other disease specifically, at least in any major way. It's far, I'm sorry. Do you wanna cross any? No, no, no, in short, no. No, I haven't seen any association with that. And so most of the GWASs, I mean, they go through all of these genes. There's multiple SNPs, so if there's any association there, I would pick that up. Jason or Alan, have you ever seen, I'm trying to think, I've never seen a pseudo exfoliative with keratoconus. Yeah, I mean, the only thing close to that would be my own patient who had, well, yes, I'll take that. Well, actually, now that you've judged my mind, is, yeah, and actually, a patient I have as a younger person who had bilateral corneal transplants for keratoconus. And I followed for, probably 15 years, but about four years ago, he developed exfoliation in one of his two eyes. But I wasn't thinking in that term, actually, Bala. That's a great thought. I was thinking more in context of he had a PK rather than a trauma PK, but he did, in fact, have keratoconus. So yes, I do have one. But I don't know what that means in out of one, right? So one of the things that we found, and I was actually not sure what to do with these young patients, but it may actually be interesting to look back. There's a few patients we identified on the database here that are very young. So, and Rand actually thought that was interesting, and we can go through the charts and see, actually, if they had trauma, if they've got some sort of corneal abnormality, and so forth, et cetera. Yeah, and so we could actually, that'd be an easy thing to look up and see because it's pretty uncommon. That would be very unusual to see them both. But the problem with the keratoconus patients is they're so young. So they'd have to be old keratoconus patients. And then, I forget, so I don't know how many corneal folks are in here, but they were saying that you don't see old keratoconus patients. They seem to, like, disappear. And I, and there's only, there's no good reason for that to happen. So we started wondering, are they dying off and no one knows it? Has anyone ever heard that? That's very interesting. You mean as far as the Icelandic? So actually higher the latitude is a risk factor. Yeah, well actually it is very prominent here. And yes, it's very prominent. And what I thought was odd was that the comment that Scandinavian ancestry did not appear to be associated. But I have to say I'm a little, I'm wondering just a little bit how that data was confirmed for one. And I still do wonder because I know here there's a lot of exfoliation. We don't have a lot of Scandinavian folks in North Carolina, we have much less exfoliation there. In our glaucoma population it may be 4%, 5%. I don't know in the glaucoma population here what percent. So quite a bit more, right? So the whole Scandinavian question, I've just kind of not paid a lot of attention to. I think it's interesting. But as far as the risk they are, it seems to be light associated. So they're, you know, they are pretty much an island and they're at a high latitude. To be honest with you, I'm not entirely convinced of that data either. Because actually if you look in Africa, you have the equatorial region and on West Africa, you literally don't see it at all. And then on East Africa in Somalia and Ethiopia, it is rampant and actually Saudi Arabia, that whole belt, there is just all kinds of very high prevalence of exfoliation. Now I mentioned that to Lou and Josh and said, well what about that? And then he said, their thoughts are well, oh but that's a desert, they get lots of sunlight. You know, it becomes a lot of hand waving in a hurry. And so I'm just, some of this stuff I throw out there, but it is curious, people get it at really different ages from 40, 50, 60, 70, 80, that there certainly are factors probably involved that are stimulating this process. A study I didn't show, Ursula did in Germany, was took fibroblast cells and exposed them to reactive oxygen species, TGF, but also just light UV exposure. And in that cell culture, that really shoots up lock cell one expression. Of course it does a lot of other things also, but lock cell one's very reactive just to light exposure. So one of the things we've been wondering is light exposure really is a trigger for this process. And then as we get older, we start shutting down our genomes anyway, and then we croak. So it's like when you start shutting down some genes, it may be that the balance factor starts really making a difference. So again, we don't know the functional piece of the puzzle yet, but we are really getting very, it's what the exciting part is, at least for us, is that we're getting very close to identifying specifically that computer code that puts us at risk for this disease. And that is just gonna be so exciting. Talk to at least 20% of the room at this point about the really cool studies that we can do with this once we know that we have that key. Well, I wanna, again, I mean I'd be happy to answer questions I don't like dragging people forever through these discussions, but I'm here, I've got lots of time today. Anyone who wants to talk to me, I really do appreciate it with you all this morning. It's a lot of fun. Really had a great visit. Thanks. Thank you very much. Thank you so much.