Chapters Transcript UBA1 Genetics Course: 2nd International VEXAS Workshop Thank you for MIU for putting this up, to have us here in this great city and great location to meet up for a 2nd Nexus meeting. Thank you, David, for the topic. I mean, what can I say about UBA1 genetics, the whole room of UBA1 experts and and uh vaccatologists. So I'll try, I, I was trying to dig out some facts which may be of fun and maybe give us some insights in terms of the disease. So we'll just take it from there. And also, I'm going to be quoting quite a few of your studies. So if I've got something wrong, please don't, you know. be too upset, and if I haven't necessarily included your study again, please don't be too upset because you only have 20 minutes to go through this. So first of all, Origins, where did we, you know, you know, first heard about the UBA1, and I found this quite interesting actually, is that we had protein first before we had the gene. And actually the protein was isolated as an enzyme that had ATP dependent erein activation way back in 1982. And it wasn't really until quite a few numbers years later that the gene was isolated and actually it wasn't the silly gene thought to be the, you know, coding for the UBA1, it was the gene that was linked with a defect in a cell cycle of cell division, and there were these really clever, it's quite interesting reading these papers how people used to do this before all this technology we have today and single cell sequencing and how they deduced all this, but basically they were Putting back in bits of X chromosome and then figuring out which bit of X chromosome actually rescued the, the phenotype, and they found that there is a bit of an X chromosome human gene that actually rescued the, the cell division. Uh, and he was given this name that was temperature sensitive gene, which turns out later on it's the same gene that calls for the UBA1. Uh, there is in the animal kingdom Y homologue. But not in humans. So in humans we don't, we lost the homologue on the Y chromosome, and also it's not a gene that is subject to X inactivation. So again, an important point, like many others, just kind of illustrating the importance of the UBA1. So in terms of then, sorry. This is a slide that you're very familiar with, but I think it was important to just put it up, just to orientate us that the UBA1, as we know it does code for two isoforms. The one nuclear nuclear one which has nuclear nuclear location bit at the beginning, and the cytoplasmic one. Now, the difference between the two is relatively small, other than location, but that's probably gonna be an important point, which I'm gonna come back to later on. And obviously, there's all these other functions that now have been linked with the UBA1, but largely really with the cytoplasmic component of the enzyme. And I think a lot of what we know in terms of what goes wrong when the UBA1 is not working properly is linked with its cytoplasmic effects, but not as much is known really what happens when the Its function with the nucleus is disturbed, and that's something that I would like to So, what I'm gonna try and do for the next several slides is use a number of human conditions where the UBA1 mutations have been linked to try and maybe tease out some of these differences and think about how, what the bits of the story we know, what bits we're not so sure, and what bits we should be maybe focusing on. And you have also already highlighted some of the bits and pieces that I'm going to be talking about. So, the first one, in terms of um The story is actually the first condition with which the UBA1 variant, rare variants were linked with this, which is this X-linked spinal muscular atrophy. And this condition was first described in 2008. Uh, and actually, when you look back the original paper, the level of mechanistic proof wasn't huge in terms of how they've connected the two. And in essence what they've shown is that yes, there were these rare variants. They somehow led to some reduction in the UBA1B expression within the cell, how that was. Why was that happening wasn't entirely clear, given that all these changes were largely SMVs. But interestingly, all these patients were male, of course, because the gene is an X chromosome, and they all presented with these largely neurological features. And there is an important point to think about, given that it seems that obviously the ubiquitination, although it's present in all the cells, some cell types are more susceptible to loss even of the small amount of UBA1B within the cell and ubiquitination disorder. And actually it's the neural cells that seem to have this particular susceptibility to even partial loss of UB1B function. And although in this condition, obviously the defect is present in all cells, it's only the neuronal cells that are affected. Furthermore, the subsequently, female only cases were also identified, and those cases. The mutation is actually much more damaging. It leads to complete loss of one of the allele functions. So therefore, even the ha insufficiency, if you like, of UBA1 in these cases leads to a form of neurological phenotype. And there is this link with aging and reduction in the UBA1 expression as we age, and that may be relevant to some of our patients that actually It's not just the mutation, but also the timing and the age of the patient that may be linked to some of the reduced activity of the UBA1. And actually what we seem to be coming back to is that a lot of the time patients may be tolerating the disease or these variants because there is some residual function which is needed really for the disease expression. So, this is the one we're all very familiar with. Something magical happens and some, you know, something leads to, obviously, and this is a bit that everybody keeps asking, why do I get these mutations? Well, I guess it's just a random event of aging. We get the mutation and the ones that we now call canonical are the ones that we link quite clearly to the UBA1 to the vaccis syndrome are the ones that affect the methionine 41 position. And there are either there are some, somebody mentioned there's splice site variants which affect, again, it's very similar principle that you lost the expression of the UB1B. But with the splice site variants, actually, you do get um quite a profound loss of, of uh UB1B expression and the pathology has been relatively well worked out, and I couldn't really resist not to put the slide in. Adam and and Daniel from Dave's lab, they've worked quite hard in producing this picture, and this, you know, this kind of very small change produces a disease which manifests in all these different shapes and forms and affects pretty much every organ system that you can think of. And why is that important? Because of what Yahi was talking about and what we're trying to figure out about this genotype genotype correlation. Can we actually guess from the mutation which one of these complications is going to happen? What is actually going to cause most of the mortality and morbidity in these patients, how we can actually prevent that? And there is some data which has now, this is, you know, given what David has shown in terms of the amount of publications now, it's dated 3 years ago. But actually this was one of the first large retrospective cohorts that was done together between here and in the in the US and we contributed data from the UK and led by Marcy, but that data first indicated there maybe was some difference in terms of genotype, phenotype, and we could predict at that point that lucin. The Davalian is probably linked with the worst outcome, and it seems like that that story keeps repeating, which is good, you know, it's kind of reassuring that we're seeing that to some degree, and obviously more prospective studies are going to be needed to fully show that. And Akim and David had this wonderful idea about why that may be, and this is really because there seems to be the level of residual UB1B function that is really relevant to why there is a difference in the phenotype and actually why is it that disease can manifest because You do need some degree of some residual ibi vaccination, I guess, for the disease to express. Without it, some of the variants which cause complete loss of translation are not compatible with the cell survival. So there's this. Magical combination of some of the UB1B activity linked with obviously reduction of the UB1 function that allows for the disease to be expressed, and that itself is also then linked to some degree to this genotype phenotype correlation. And that this is now from other retrospective studies largely and from France and some blood from Sophie and others from Frembek's cohort that's shown that there are these correlations that seem to be holding for a while until we have, I guess, major international consortium which pulls together all these patients and we find that maybe this is all nonsense. Hopefully not. And then um was mentioned, obviously, and we all talked about from the beginning. Is this important? How much mutated cells are there? Is it important in terms of disease expression? What happens to the patients and so on? And this is a study that David and his team published recently going back to that original cohort. Geisinger Health where they revisited those patients and looked for using the muttech 2 analysis to look at for very low level variants that were not necessarily picked up during the original analysis and in essence what they've shown. Is that yes, you can actually link some disease progression with increasedwa and actually some mild disease activity may be present and actually macrocytosis is something also that evolves over time and might not be present at the beginning when the VF is low. But actually, in the next slide, I'll mention also some of the study that Peter did in terms of the other cohort. Which again shows the same principle that maybe LAF is not always disease penetrant. There has to be some magical level and it depends really on the mutation, what that level is before you can have disease expression. So this is, you know, bringing us back to the epidemiology, how a common vexes is in terms of, you know, if we base our assumption that these mutations are all highly penetrant and, and, and cause, can cause the disease, and clearly again, going back to the Geisinger cohort in the study that they did a few years ago, showing that, yes, and we had these conversations. I remember when it was first described like how common do you think this is going to be. Well, at least looking at my clinic cohort, I thought it's going to be common. And actually, you know, it was shown in the study that it can be as high as 1 in 5000 men over the age of 50, and in repeat study of that cohort, probably roughly those figures were were repeated again. But then the study, the second study from the US which looked at completely different cohort of patients which included much more healthy individuals, not necessarily individuals who will As for uh uh were engaged with the healthcare, show that this is maybe not the same, uh, and here there was quite a lot of females may all interestingly all had lucin variant, and what also and and and the men within this cohort, which were few, had a very relatively low of this lecin variant and actually, There was no documentation in the clinical records of any symptoms or causes that would suggest a disease that may be linked to what we understand to be vexes. So again, suggesting that maybe lova, particularly with some of these less pathogenic variants like Lucy, may be tolerated for longer. And we did something similar in the UK. We've used several different cohorts to look again to see how common vaccine might be, what populations we should be concentrated on, and so on, and we looked back and looked into the UK Biobank. This is basically a study which has been going on for a number of years. There's 5, half a million participants in that UK biobank all had either whole exome, whole genome sequencing, and essentially we only managed to find one canonical mutation in all that population. Which kind of made us think like why is it that we haven't seen more? And again, this is a different population. There are many more younger people included in there and so on and so on. But actually, if you focus on those patients who are being investigated for unexplained anemia over the age of 15, if they're a man, in that population, actually there is a significant enrichment for potentially new cases. So we found that about 171 new cases in England which Not the whole UK, but England is about 50 million people. You, you, you would expect to see 170 171 new cases of access in men over 50 who have been investigated for some form of cytopenia. And then, you know, again, it was questioned very early on, you know, is the vaccus the disease of vast or is it found anywhere else in the world and so on, and, you know, Sophie and Tim and David again looked at through the literature and obviously shown that as as the vaccine has expanded and the knowledge of the vaccine has increased and awareness has become greater, people have been looking for it and clearly it's not just in Europe or in the US, it's everywhere. And I'm sure there will be many more cases in many other countries that, you know, are waiting for their first vaccine declaration. And uh, you know, it, it's great when people find this diagnosis. It's quite rewarding to actually finally get, get diagnostics. And then of course, we talk about vaccine being largely disease of man, but let's not forget there are female cases, and again, So far in the literature, most of the female cases have been those who are X chromosome deficient for whatever reason, and actually in terms of their clinical manifestations and presentation, they don't look that different to men in terms of the disease features. Interestingly, we did find one female case who was had a completely normal ch chromosome numbers. There was, and there was no, and we also looked for X inactivation, which wasn't evident at all, and that person, you know, she suddenly died. It was done from a retrospective cohort but actually looking back through her notes, she had a phenotype which completely fitted with Wexer's syndrome. So, Unfortunately we weren't able to go back and study that further and to understand really why and how Max's disease was manifesting there, but it's an interesting one to consider. And then we, there is a lot of now talk about non methanin 41 non-canonical variants and clearly what do they do and why they're important and are they important in the first place. And we were lucky, you know, very shortly after vaccus was found, we found this case of somebody who was actually screened initially for vaccus, but he didn't have a methan in 41, we found this. Serum 56 variant and with Akeem's and David's help managed to do some functional work showing that basically this variant does have a functional effect. Interestingly, not at the uh it's only on the physiological temperature that it does have reduced effect on ubiquitation. And this is not the first patient. Subsequently we found a whole group of these cohort of these patients with exactly the same variant. These patients don't have any other MDS defining mutations, but they do present completely differently to classical vaccine, and they're more like MDS. They present with much more produced profound pancytopenia with very few inflammatory, any complications, and obviously there's that French. Uh, paper that showed the other, uh, uh, uh, similar finding in, in a different variant of focusing on adeination domain of, of the gene. Again, again, showing a different phenotype, which is largely cytopenia, not, not necessarily inflammatory. And again, from the similar study that we've done looking at the canonical mutations, we looked at all these other rare non-canonical variants, and there are quite a few of these, some of them are clustering in a functional domain, some of them are repeated and found in more than one patient with MDS and so on. And then our question obviously remains, how they, do they cause disease? Do they contribute to the pathogenesis and what their role is? And some of the early insights into this again has come from David's and Kim's work showing that obviously some of these non-canonical variants can affect other stages of ubiquitation, not just the initial part, and this work is ongoing really to understand the effects and in particularly different compartments. But I like this slide and it brings me back to Um, the original kind of discovery may be that, you know, maybe this is to do with the nuclear function of the UVA1 because obviously the canonical mutations really only affect, mainly affect the cytoplasmic UBA1B isoform, but the non-canonical mutations affect both. I mean, the enzyme activity UBA1A and UB1B is equally affected. So, and it might be that there is a difference here between, and this is the nuclear. There will be of course effect on both, but the effect on cell division, which is probably what maybe is driving the cytopenia in these patients, is what is the cause of all of this, and that remains subject for further studies. And then just to finish off, you know, looking for, what else does the UBA1 do and where we can find it in any other situation. So actually UBA1. Has been thought about a lot in solid organ malignancies and actually it was noted a long time ago to be a potential target for new cancer treatment, and that's because it's highly expressed in cancer tissues and actually it's got negative predictive value as a biomarker and it was thought to be a good target because if you can reduce the ubiquitination. You can potentially induce cells to die quicker, I mean, as you would expect. So a combination of chemotherapy agents and then reducing the ubiquitation, the chemotherapy effects may be having a better, more profound effect, and that still remains actually under exploration as a potential target for some of the new therapeutics. But interestingly, there are some somatic mutations which have been found. In lung cancer of never smokers, which have shown to be enriched there again, what the function of those mutations is, it's unclear. So to conclude, you know, germline mutations associated with XLSMA. have been shown to cause this mild moderate reduction in UBA1 function, and this is why they may have this very selective tissue involvement and may be telling us something about the wider role of UBA1 in other tissues and maybe held some relevance to vacces at some point later on. This penetrance of the canonical commutations, although initially we felt they're all highly penetrant, there may be some. Obviously it's not that straightforward. Clearly depends on the type of the which somatic mutation we're talking about and maybe the level of vari variant that has been discussed during the end of the last tour because the vein, the lower buff might be more more penetran than lucium and so on. The methionine 41, non-methione 41 mutations, I think, you know, this is the new, this is the new frontier now to really look for those things, what they're doing, particularly the isophone specific function. Clearly there will be, you know, all these new discoveries that may be in completely different disease states that are not vaccine. And I would like to again, thank you for listening, and this is uh Leeds group having a bit of a rest from the vaccus. We never really rest from excess, but you know, on that particular occasion there was a few drinks celebrating this paper that was published in the British Journal of Hematology, and thank you for your attention. Published September 5, 2025 Created by
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