I am Nanda Kerkar from the University of Rochester Medical Center in New York, and my co-moderator is Jamie Chu from the Icahn School of Medicine, Mount Sinai, New York. And I think with that, we'll get started. Our first speaker is going to be Dr. Valentina Medici from the University of California Davis, and she's going to be talking to us about primer on copper metabolism, ATP7B, and epigenetics. Thank you so much. Good morning, everyone, and I would like to thank the organizer and the moderators for inviting me here today. It is really a treat for us in the field to be pretty much on the first day of the liver meeting and presenting on Wilson disease. So these are my disclosures. I'm a professor of medicine at the University of California Davis, a hepatologist and a physician scientist and a clinical investigator, and very much involved in the clinical and transitional research training for our graduate students and postdocs. So first of all, I will start with my main message, that Wilson disease is not just a disease due to mutations of the ATP7B gene. We do have some established knowledge that it's a disease of copper accumulation in the liver and brain. Copper metabolism is highly and tightly regulated. There are hundreds of known disease-causing mutations, ATP7B disease-causing mutation, and some of them are epidemiologically and mechanistically also well-characterized, including, for example, the H1069Q missense mutation, which is more prevalent in the populations of Northern European and Eastern European descent, or the R778L mutation, which is more prevalent in the Asian population. But the matter of fact is that there is no convincing genotype-phenotype correlations, as been shown now in thousands of patients. And it's possibly also a disease of no-without full penetrance, meaning that the genetic prevalence is probably higher than the clinical prevalence. With that said, I will take a dive into the copper metabolism. So although we tend to give a negative connotation to copper metabolism, copper is an essential mineral nutrient with oxidation-reduction properties and both beneficial and potential toxic effect to the cells when it accumulates in excess. Copper is present in the diet, Western diet, it is abundant in shellfish, nuts, chocolate, and of course also in the liver for the ones who eat it. And here is copper metabolism in the body. First, a general overview. In the diet, with the diet, we absorb or we receive about between 2 to 5 milligram daily of dietary copper. About half of it is lost to the GI tract, and about half of it is absorbed in the portal tract. Once in the liver, it participates to metabolic processes, and then it's released in the systemic circulation, primarily tightly bound to seroloplasmy. And only a small portion of copper is actually secreted to the kidneys, and the remaining is re-circulated in the biliary tract and eliminated again through the stool. So a little bit more in detail, again, zooming in, the copper is absorbed to the copper transporter to the small, primarily to the duodenum and the proximal part of the small intestine, to the copper transporter CTR1. Then it's released in the portal tract through the ATP7A, through the basolateral membrane, and it's loosely bound to albumin, which carries copper to the liver. Then dietary copper is primarily released to other extra hepatic organs, where primarily it's needed in the bone marrow, skeletal muscle, in the heart, and in the brain. When ATP7B is mutated, copper will not be integrated into seroloplasmy, and of course it will accumulate in the liver. So therefore, copper cannot be created and cannot be destroyed. And the copper metabolism is tightly regulated and essential for cellular metabolism maintenance. And there are two, essentially, major pools of copper. Most of it is actually tightly bound to seroloplasmy. And then there is a portion of copper that it's pretty much a matter of discussion in large studies nowadays, that it's laval copper or non-seroloplasmic bound copper, that it's loosely bound to albumin or other proteins for metabolic exchanges. So in the hepatocytes, copper metabolism is fairly complex, but also well characterized. So when it's absorbed in the hepatocytes, copper is delivered to subcellular organelles, primarily like mitochondria, but also other transgolgi network and antioxidant enzymes, where essentially where copper is needed. When the ATP7B is mutated, copper will not be integrated into seroloplasmy. There will not be the production of holoceroloplasmin from apo-seroloplasmin through the maturation to the transgolgi network. And eventually, it will not be excreted also in the biliary tract. Will first initially be bound to methionine proteins, but also accumulate in the lysosome, and eventually will be free in the cells to cause oxidative damage, oxidative stress to proteins and even DNA. So with that said, to add complexity, the ATP7B gene and proteins are also complex themselves. So the ATP7B gene, it's a large gene based on 21 exons, where you can see here in the red boxes, most of the mutations, the missense mutations are in the exons 8 and 14. And a transmembrane copper transporter, it's a transporter with 8 transmembrane portions, and the N-terminus portion bind copper. And on the other side, there is ATP binding sites for the active transportation of copper outside the membrane. So with all these complexity in the genes, the mutations that can affect the genes, the metabolic mutation, the copper transporter, well, there is no convincing correlation between genotype and phenotype. And Wilson disease has been described as a great masquerade, and rightfully so. So I spent the past 15 years trying to figure out the relation between the extra hepatic ATP7B, between genetic factors, metabolic factors, and also ultimately epigenetic factors, to try to explain the phenotype of this disease. So what we started from was the DNA methylation mechanism. So obviously, one of the best characterized epigenetic mechanism of gene expression regulation, through which methyl groups are covalently bound to cytosine bases, to modify the accessibility of the DNA to transcription factors, and consequently modifying the transcription potential. And we started working on this, and not just by chance or because it sounded good in Wilson disease, but it's because copper heavily affects this metabolic pathway, that it's a methionine metabolism. And why is that important? Because methionine metabolism resides at the interface between nutrition and environment, if you want, and epigenetic regulation of gene expression. In particular, copper, when it's in excess, can inhibit that enzyme, that it's indicated here, that it's S-adenosyl homocysteine hydrolase, that it's bidirectional enzymes that metabolizes S-adenosyl homocysteine to homocysteine. So when it's inhibited by copper or other factors, we can speculate that the upstream S-adenosyl homocysteine will accumulate, and itself, it is an inhibitor of DNA methylation reactions. So what we studied, what we started working on mouse models, both working on the THJ mice, which present a spontaneous mutation of the ATP7B copper transporter. Also we worked on the ATP7B knockout mice. And we then translated our results to patients with Wilson disease, and then again validated mechanistically the results in the mouse models. With that, we started with this study, now several years ago, and with this evidence and also other evidence showing that immunofluorescence for 5-methylcytosine, so just an indicator of global DNA methylation in the nuclei of the hepatocytes from THJ mice, the signal was significantly reduced in the animal model of Wilson disease compared to mice with normal copper metabolism. And when we provided beta to these mice, so the methyl group donors, the signal was kind of catching up and becoming back to normal to the signal of 5-methylcytosine and the indication for global DNA methylation like in normal mice. So with this evidence and other evidence in mice, we concluded that methionine metabolism is heavily dysregulated in animal models of Wilson disease. That is probably due to copper effect, the inhibitory effect on the hydrolase with the upstream accumulation of S-adenosyl-homocysteine, and ultimately resulting in global DNA hypomethylation in the hepatocytes. With that, it was our time to translate our results to patients with Wilson disease. So we conducted the first whole-genome bisulfate sequencing in the liver and blood of patients with Wilson disease. So with this analysis, it's a relatively unbiased approach to study the distribution of methyl groups throughout the whole DNA, including exons, introns, enhancers, promoters, and compared these to other healthy subjects and other patients with other liver diseases. And the results are expressed in percentage of different methylation throughout the DNA. So as you can see here, so essentially we compared. So this is the first representation of the differential methylation in the liver of patients with Wilson disease compared to healthy subjects and compared to patients with other liver diseases. And those included autoimmune hepatitis, fatty liver, and also primary sclerosing cholangitis as a model of copper accumulation. Of course, not as much as Wilson disease and obviously a completely different mechanism, but also it was a kind of a positive control. And you can see here in the red circle the difference of the hypomethylated and the hypermethylated differentially methylated regions that are distinguishing Wilson disease compared to healthy subjects and other liver diseases. So we identified more than 900 hypomethylated DMRs and more than 800 hypermethylated DMRs separating and characterizing Wilson disease. And we did the same exercise in the blood. So obviously the blood less, a smaller number of differentially methylated regions, but again almost 200 hypomethylated DMR and 75 hypermethylated DMR in the blood. Obviously the blood is expected to have less genes because it's typical of Wilson because characterizing Wilson because it's affected by obviously so many organs and factors. And what we did next, we overlap the findings in blood and liver. So to characterize that signature that in the blood, methylation signature could also represent what was happening in the liver. And so we identify a smaller number, of course, but still of hypomethylated and hypermethylated differentially methylated regions that could distinguish Wilson disease both in the liver and blood. And with doing that, we identify a number of target genes that have a potential pathogenic significance. And the first one that we focus on and I will talk about today is the histone deacetylase 5 or HDAC5, which was significantly hypermethylated with similar direction in both in the liver and blood of patients with Wilson disease. And this was a very interesting target because then we confirm from humans to mice that HDAC5 levels were reduced in multiple models of Wilson disease, mouse models. And it also was progressively reduced, worsening with the progression of liver disease and progression of fibrosis. And also levels were changed by co-percolation and by the availability of methyl groups. And this was a particularly interesting target for us because, or for the pathogenesis of Wilson disease, because these enzymes, so histone deacetylases, remove acetyl moieties from lysine residues of histone tails, therefore modifying the transcription potential. And HDAC levels are strongly influenced by nutrition factors, therefore again laying at the interface between nutrition and diet potentially and epigenetic regulation of gene expressions. And particularly HDAC5 is important because it has, can affect the expression of genes fundamentally involved in lipid metabolism and cell proliferation. So we confirmed that HDAC5 is reduced, the signal is reduced in the nuclei of the hepatocytes of a model of Wilson disease. And to identify the genes that are regulated by histone acetylation, we conducted and integrated the first analysis of H3K27 acetylated as a major marks of histone acetylation, previously studied in other liver diseases. And we overlapped results of chip sequencing analysis for H3K27 acetylated and RNA sequencing, identifying co-upregulated genes and co-downregulated genes that are dysregulated in liver or liver of mice, TXJ mice with Wilson disease. And conducted the pathway analysis on these genes, identifying how the pathways, the genes affected by histone acetylation were fundamentally involved in pathways of lipid metabolism, fatty acid oxidation, and cell metabolism in general. And we did the same analysis also regarding on gene transcription factor genes and identified some major transcription factor regulated by histone acetylation, including PPAR-alpha and PPAR-gamma. So this is our working model that we published a few months ago, showing that, yes, Wilson disease start with copper accumulation and oxidative stress in the liver, that it's associated with changes in DNA methylation, that also affected the expression and levels of H-ducts or histone deacetylases, which affect on downstream marks of histone acetylation. And those affected the expression and regulation of major metabolic regulators and transcription factors, including PPAR-alpha and PPAR-gamma. So my key messages is that Wilson disease, likely its phenotype, is affected by epigenetic mechanism at the interface between DNA methylation and histone acetylation. And these have likely consequences, like how the environment, and especially diet, can affect possibly the phenotype, how the phenotype of Wilson disease can take the direction of either neurological or hepatic predominant presentation, how eventually this can affect the response to the entire copper treatment, and potentially also the response to gene therapy, which might be the future. And well, I would like to thank you for your attention. Okay, thank you so much, Dr. Medici. Our next speaker is Dr. Benita Kamath, who will be talking about Could This Be Wilson Disease? Diagnosis and Management in Children. Thank you very much to Dr. Chu and Karkar for the invitation and to ASLD. So this is a vast topic for 18 minutes, so I hope that you will indulge me. This is going to be an overview and hopefully just a practical approach to what we as pediatricians should be considering in the diagnosis and management of Wilson disease. These are my disclosures, none of which are relevant to the talk today. This is just an outline of what we're going to go through, the presentation in children, diagnostic approach, highlighting the new practice guidance that we will hear more about, I'm sure, today and in the meeting, histopathology, a little bit about the approach to mutational analysis, scoring systems, and treatment in children. So I'm going to start with a case. Could This Be Wilson Disease? That was the title I was given. So this is a 10-year-old girl who had an incidental finding of elevated transaminases, fairly typical. Total bilirubin of zero, didn't have to put in the units, that was easy. Transaminases ALT of 169, slightly elevated GGT of 80, and an ALKFOS of 329, and this is a real case. Physical examination was unremarkable, her BMI was 85th percentile, possibly normal for the U.S. I'd consider that generous in other parts of the world, but nevertheless, and her ultrasound showed some mild diffuse increased glycogenicity and a suggestion of common bile duct thickening on the ultrasound, common bile duct wall thickening. I was thinking, oh, possibly early PSC, and then we did a, followed up with an MRCP, which confirmed a mildly steatotic liver and a normal biliary system. Okay, so could this be Wilson disease? Well, let's look and think about the literature. So, oh, sorry, a couple of additional things about her case. She had a slightly low borderline alpha-negative trypsin level, unremarkable serologies, normal IgG, and ANA was positive 1 in 640, and a serolaplasmin was 229 in our units in Canada, which is equivalent to 22.9 milligrams per deciliter. Right, so we'll come back to the case later. So what about the presentation of Wilson disease in children? We know it's predominantly hepatic, and I'm going to focus on that today, knowing that we'll probably discuss more about the neuropsychiatric manifestations later. It is increasingly being diagnosed in children under the age of five, so I do think we have to shift the dogma that perhaps that we had in pediatrics, that it's really a disease of, that we should only see it in school-age children. And neuropsychiatric symptoms, although they're rarely seen in children, especially under the age of 10, we do still think about it in our older groups, such as, and we have classic, we can see classic motor, psychomotor incoordination, declining school performance, tremor, but sometimes the features can be quite subtle in children, and can manifest as behavioral issues and mood disorders. So it is something to be alert for, and I think it's sometimes something that we can uncover after we've recognized the hepatic presentation. So focusing specifically on the hepatic presentation, I like the idea of thinking of it as a masquerader or a mimicker, as we heard earlier, because it just has such a diverse presentation. It can be asymptomatic, truly asymptomatic, as we see when we do screening in first-degree relatives, or the features are just subtle and unrecognized. We can even just see isolated splenomegaly. The classic presentation that we see, I think, most of the time now is biochemical abnormalities, and a presentation, and this is really key, that mimics autoimmune hepatitis or fatty liver disease, and that really is probably the most, I think the most common presentation that we need to be alert to to make sure that in that setting, we are not missing Wilson disease. Cirrhosis, of course, important, compensated and decompensated, and then classic Wilsonian acute liver failure in the setting of non-immune hemolytic anemia, relatively normal transaminases, and very low ALKFOS. And acute liver failure in Wilson disease is a whole talk all by itself, very interesting. There's multiple case series of these, hard, I think, to think about in pediatrics because we have such small case series. We, a few couple of years ago, did a meta-analysis of this in 256 patients, and the clinical characteristics of these patients was interesting, I think, to me, because you see a lot of Kaiser Fleischer rings, which we don't typically see in our classic biochemical abnormalities group that we see most of the time, a lot of the anemia, and also 78% of that group also had encephalopathy. And this is a meta-analysis, so it suffers from the limitations of all meta-analyses, but I still think it gives us a nice view of what pediatric Wilsonian acute liver failure can look like. And in this meta-analysis, we saw 11% spontaneous recovery. I was surprised. I think that's high. The remainder of patients underwent liver transplantation or die, and I do accept that there could be a reporting bias because lots of people were reporting the use of extracorporeal systems, but still a way of thinking about acute liver failure in the pediatric population. So that's the presentation. How are we going to approach the diagnosis? So this is taken from the new practice guidance led by Dr. Shelsky and Eve Roberts that has just been published and will be described again later in the meeting in more detail, but I've selected from the new practice guidance the algorithm that I think is most relevant to our pediatric presentation of Wilson disease. So that's a patient presenting with liver disease and absent KF rings. And in this setting, if the seroliplasmin is low and a 24-hour urine COPA is high, and we'll talk a little bit more about those in detail, you can say that the diagnosis of Wilson disease has been established, genetic diagnosis is recommended, and neurologic evaluation is considered. It's interesting that you don't necessarily have to do a liver biopsy. We can talk about whether us as pediatricians would still want to do one, and perhaps we can be dissuaded from that now. I think in my practice, I typically still do do that, and we'll talk about that later. But that's the sort of easy, the easier one where it all falls into place. So what about when the seroliplasmin is low and the 24-hour urine or COPA is also low or when there's an intermediate level? So that's when you would think about progressing to a liver biopsy. And again, with a liver biopsy, in that setting, if the liver COPA, the parenchymal COPA concentration is low, you probably are not dealing with Wilson disease here, and that you can just probably go on and look for other things unless it's a heterozygote, the patient's heterozygote. When the urine COPA is high, also relatively straightforward, you then establish your diagnosis of Wilson disease, and if it's intermediate, then you're going to probably move on to genetic testing. So I think this gives us a nice overall. Of course, there are so many nuances. I would really urge everyone to read the practice guidance. It's excellent, and it really addresses some of the subtleties and nuances when people don't fit into these categories cleanly, but this is a very nice approach. Now the red numbers in the brackets after all each of the steps here are the numbers which are linked to the Leipzig score, which is also a valuable diagnostic scoring system. This has been validated in children by Anil Dhawan and others, and so I think it's important to have this. Particularly, I think it's helpful when we're thinking about the atypical cases. And using the Leipzig score, a score of four or greater gives you an established diagnosis of Wilson disease. So just delving a little bit, thinking a little bit about just a couple of the features that were in that diagnostic algorithm, thinking about the ceruloplasmin, we know that if you have a modest reduction in your ceruloplasmin, you really still need to keep thinking about Wilson disease. Up to 20% of patients can even have a normal ceruloplasmin, and there are a host of other conditions in which ceruloplasmin can be low, which are protein-losing conditions. It's also an acute phase reactant, so it could potentially be increased in inflammation. The 24-hour urine copper, not always exactly straightforward to do in children, because you've got to make sure that they're actually going to do it. It's even been hard to do in children when they're in the hospital. I find it valuable to measure 24-hour urine creatinine at the same time to assess adequate urine collection. You can use a penicillamine challenge as an adjunct. I don't think it's mandatory. And although the normal range is, as we would say in Canada, 0.6 micromoles per day, but I've shown the others, most of the patients, the Wilsonian range is usually greater than 1.6 micromoles per day. So the role of liver biopsy. So I showed you the algorithm where, if things are fairly classic in terms of the ceruloplasmin and a high 24-hour urine copper, a liver biopsy isn't mandatory. I think that's fair. It's not mandatory. I do think that in pediatrics, we still have a tendency to do it. It can exclude other diagnosis. It can potentially help stage fibrosis. And the key for us as pediatricians is, obviously, when you're going in, you don't know that it's going to be Wilson disease some of the time. And so I think what's really important here is that we have to think about doing parenchymal copper concentration, getting that second pass on the liver biopsy in many children that we are sending for a liver biopsy. So when you think it's autoimmune hepatitis, and when you think it's NAFLD, should we really be thinking about sending that second pass? Now, in the olden days when we all did our own liver biopsies, that was up to us. Now it's really a question of making sure you send that instructions to image-guided therapy or interventional radiology, whoever's doing that, and make sure that second pass gets sent, and make sure it gets sent and is processed appropriately by pathology. So when you get the liver biopsy back, so remember, staining for copper isn't always incredibly helpful. It's great if you see it, but it's not diagnostic because even in nodules adjacent to each other, some may stain for copper and some may not. However, I really like histopathology. It's been described before that light microscopy can be overlapping with autoimmune hepatitis and NAFLD in pediatric Wilson disease, and these are two cases of Wilson disease where really you can just see portal inflammation, you can see fat, it's indistinguishable. So we looked at the hepatic ultrastructural features which distinguish pediatric Wilson disease, and this has been described before, and you can see really these are beautiful examples of mitochondrial changes. So up in the first panel, you can see dilated tips of CRIS-D, you can see pleomorphic mitochondria in panel D with different shapes of mitochondria. Down in the bottom panel, you can see fat, and then you can see paracrystalline inclusions. So really quite classic features, and we quantified these and compared them. We had three blinded pathologists review them and compared them to biopsies with AIH and NAFLD and Wilson disease. And what we've got this panel here really shows is just the clustering and the predominance of the number of mitochondrial abnormalities that you can see in the Wilson disease liver biopsies. So, and of all the features that I've just described, dilated tips of CRIS-D had a higher sensitivity and specificity. So in my mind, electron microscopy examination is really essential in the diagnostic workup of a pediatric liver biopsy. So how about when are we going to do mutational analysis, and do we need to do it? I showed the algorithm from the practice guidance. I think the one key thing, what I like about this figure at the top, it basically shows us, it gives us a guide to when we should be doing mutational analysis in association with a Leipzig sore, which you can also just take as saying, you need to have a clinical suspicion for Wilson disease before you do the genetic testing. It's not a screening test. You have to suspect, you have to have some clinical suspicion there's liver disease before we do that. So really, if your Leipzig sore is zero, should you really be testing at all? It's really valuable when you suspect Wilson disease. it's very valuable when you have atypical cases. It's clearly essential when you're doing family screening because then you can just check for the variant that's already been identified in the proband. And I certainly do like it for confirmation of diagnosis, though I will say in Canada we have an incredibly diverse population, and I frequently find mutations or variants that have not been identified before, but I think that's important for us to amass our knowledge of variants in different populations. This paper was already alluded to before that it's great to collect information about variants in Wilson disease, but is this actually going to help us with any type of prediction of the phenotype? So based on this study, which was done in really all Caucasian patients, I think there were only eight Asian patients in the entire cohort. It's a massive cohort, which does include 700 children, so it is relevant for us to think about, and really most of the patients had a very similar genotype, but there was no correlation in this study between genotype and liver disease severity. One interesting thing I think taken out from this massive cohort was just to see the prevalence of cirrhosis increasing with age, as we would expect, only 7 percent at the age of six and 49 percent by the age of 16, and as is well established, the hepatic presentation, including for hepatic failure, was more common in girls. Now it has been suggested that truncating mutations can be associated with earlier onset of disease when there's absent or non-functional protein. I just still ask myself about the effect of other genotypes. Have we fully explored genotypes in other populations? I think epigenetic mechanisms, as we heard, are also going to be incredibly important. So I still think that I – certainly the data to date suggests that there is no effect of genotype on phenotype, but I hope that we – that there's still more for us to do and that we might still be able to have some answer, because we've amassed such an incredible array of genetic information. It would be lovely to see that it could have some clinical impact as well. We're quite interested in this in Toronto, and we're looking at the effect of ethnicity on Wilson disease, and we have a poster, which I've just highlighted there, that people might want to come and visit during the meeting. So we've talked about diagnosis. Just a quick word about prognosis. This is the WD index, which was modified from adults to pediatrics so that it can be used as a scoring system to assist with thinking about which children will need liver transplantation when they present. This is the same adult WDI, but then modified by Anil Dhawan to incorporate the white count and the albumin, which really incorporates features of acute on chronic liver disease. And a score of greater than 11 predicts the need for liver transplantation. And this year, they published a study where they validated this in another 52 children and saw that an index of 8 to 10, that patients that present with that score also need quite close monitoring, as they may require liver transplantation up to two months after presentation. So we've talked about diagnosis, biochemistry, histopathology, scoring systems. Let's go back to the case. So I mentioned that she had a slightly borderline alpine antitrypsin level. She was an MS phenotype, so that's not really causing her disease. I wondered if she was a lean naffled, maybe 85th percentile is that lean, I'm not sure. And then the family told me that they live on a mushroom farm and they drink well water. I'm not making this up. This is really true. And I thought, huh, OK. So we got everybody in the family, had all the family tested. We did 24-hour urine copies in all the family, and we tested the well water. The well water was actually fine. And then the family told me that she doesn't like mushrooms. OK. But her 24-hour urine copper was actually very high, and we did a liver biopsy, which was pretty classic and diagnostic for Wilson disease. I wondered if she was a heterozygote, and perhaps, because she actually has one known pathogenic variant, and then she has another variant in her ATP7B, which has not previously been reported, but in all the prediction tools I can find, is predicted to be pathogenic. So I don't think she's a heterozygote that's exposed to copper. I think she does have Wilson disease. I just think the mushroom farm is a red herring. But it certainly led us to the right path. So just a couple of moments about management. I won't go into this in a huge amount of detail. I think this is, again, taken from the practice guidance, but it should be familiar to most of us. The mainstays of therapy are low copper diet and then zinc or chelation, or zinc and chelation, depending on your preference. I think in pediatrics, a lot of us use zinc in combination with chelating therapy with d-penicillamine or triantine. I think we all know the challenges with penicillamine. Tremendous number of side effects, early fever, hypersensitivity, rashes, and then later on set with nephrotoxicity and bone marrow suppression, all of which really make it a problematic drug. I have preferred in the past not to use it at all, but now, with the change in costing of the drug in Canada, we now have to show that a patient fails penicillamine before we can use triantine. I don't have the time to go more into the therapy here, but I think most of this and the dosing is just up there for reference. But what I did want to mention was the role of vitamin E in antioxidant therapy. And I've been very fortunate to have some training from Eve Roberts, who really helped me think about patients who have mildly elevated transaminases despite being on adequate chelation therapy, who still have and really benefit with a little bit of vitamin E. And this has been proposed as an adjunctive treatment because of the antioxidant effect. We heard about oxidative stress within hepatocytes, and we don't have sufficient data for it to be a standard of care, but I really have found it valuable, and I think that there are clinical data that support it, and the end point for using it is improved aminotransferases. The monitoring of kids, I think we know, with the chelators and zinc. Just for time, I'm not going to go into this into more detail, but I think it's important to look for clinical improvement, biochemical improvement, and, of course, a 24-hour urine copper, which initially you were going to do every few months, and then at least once a patient's on maintenance therapy, to think about doing it at least annually. Just a quick mention of non-ceruloplasmin-bound copper. Can be useful, shown here. When it's less than five, it's suggestive of overtreatment. When it's greater than 15, suggestive of non-adherence, but I will comment that, depending on the assay that you have in your institution, if the whole of ceruloplasmin is overestimated, you can get a negative result, which then limits the usefulness of this. So, finally, quick note about pre-symptomatic and very young children. I think the key is, this is useful for screening for asymptomatic individuals. The key is to look for the extent of organ damage before you think about treating, especially in the very young and in the truly asymptomatic, and this may be a scenario in which I think monotherapy is appropriate. Just one quick note, I think, which is interesting, is that thinking about newborn screening in the future may be an option with ATP7B proteomics, which may be coming in high-risk populations. So my high-risk, my take-home points are that consider Wilson disease in every child with NAFLD and AIH. Hepatic presentations dominate in children. Liver biopsy, I still think, is really valuable, and we should be thinking about it when we don't think it's Wilson disease, and there's still an important role for mutational analysis and diagnosis. Thank you. Thank you for a wonderful talk, Dr. Kamath, and I'd like to welcome the next speaker, Dr. Fred Ascari from the University of Michigan, who will be talking to us about the diagnosis and management in adults, including pregnancy. Thank you so much for inviting me to present. Obviously, I'm talking about the management in adults to a group of pediatricians, so we'll try and point out some contrasts as well as we go through this. These are my disclosures. Wilson disease therapies, I'm going to talk about presentations and diagnosis, the evolution of therapies, current medical treatments that are available and their advantages and disadvantages and challenges, and I'll briefly touch on transplant as one of the other therapies that's used. So, Wilson disease was first described in 1912, as you know. The Copper Association didn't come on until 1948, and shortly after, people started using BAL, a British anti-lewis site agent. Penicillamine was approved by the FDA as first-line therapy in the 50s and 60s. Triantine was approved as a salvage therapy for those who fail penicillamine, as the previous speaker discussed all the tragic side effects with that drug, and zinc was first studied in the 60s in the Netherlands and was approved by the FDA as maintenance treatment in 1997. So those are the currently available treatments. In terms of copper toxicity, we're talking about a spectrum of liver histology ranging from normal histology to mild inflammation, steatohepatitis, glycogenated nuclei, cirrhosis or liver failure, basically acute liver necrosis. We have neurologic movement disorders, which we see more in the adults than in the pediatric population, although some children do get them, as well as psychiatric illness, and then a presentation diagnosis by an ophthalmologist or optometrist of K.F. rings. So those are some of the impacts of the copper toxicity. In terms of diagnosis, the previous speaker went over the Leipzig score and the ASLD guidelines. I just kind of quickly review that the diagnosis is made using these parameters, the urine copper, the ceruloplasmin, the serum copper, the liver copper quantitation from a liver biopsy, histology, which can be nonspecific, but we want to be sure it doesn't show cholestasis. Obviously if a biopsy shows PBC or evidence of small duct PSC or biliary obstruction, that would be a problem in terms of interpreting the liver copper quantitation, K.F. rings. MR imaging shows copper damage rather than copper. MR basically is useful for seeing ferromagnetic elements, but it's not useful for copper, which is non-ferromagnetic, but we can see the damage in specific areas of the brain or see damage to the liver that suggests they might have Wilson disease or some other damage. And then obviously gene testing has been talked about. What I'm not going to talk about much is this calculated non-ceruloplasmin copper because it's often negative. It's fraught with measuring and calculation areas, and the field is likely to have a funeral for it. I think the next speaker may talk about some of the, you know, more emerging diagnostic testing that we hope will replace the non-ceruloplasmin copper, but I just point that out because a lot of the prior guidelines and guidance and old teaching, you heard this used a lot and you noticed that we're not talking about it much, so I'm actually going to talk about it to say I'm not talking about it. The clinical recognition is obviously a major problem. There are types of presentations. Obviously in the adult, we see really a broad spectrum and combinations of neurologic, psychiatric, hepatic, asymptomatic, and then what I would classify as other presentations, like I mentioned the KF rings. Family screening is really important. Obviously every sibling has a 25% risk of having Wilson disease, and so really we want to pick up those cases, and that can be very challenging to get them to go do screening, but I do want to emphasize that. Obviously genetic screening, we're seeing more patients come in, our obstetric colleagues are really pushing prenatal genetic screening, and we're seeing more mothers coming in with I've got Wilson disease to our clinics from this, and obviously finding out about kids who are going to be born that have Wilson disease, as well as birth cohort screening, so that's already being used in the state of Washington. Siyon Han has initiated this, and we're going to see, hopefully this will become largely a pediatric disease that transitions to adult hepatologists rather than right now where we're seeing many adults leaking through without the diagnosis because they haven't gotten sick while they were kids, but I think that's an important presentation. So in terms of neurologic diseases, we think about this as a movement disorder. Tremor, primarily resting in intention, is a very common finding. Sometimes the patient can present with dysarthria, trouble talking, slurring their words, or having bradykinesia, slow speech. Trouble swallowing, which as gastroenterologists, those of us who may see occasional GI patients, we'll see them coming in complaining of dysphagia. Dystonia is obviously an issue, as well as bradykinesia, gait disturbance. The classic wing-beating tremor that's described in Wilson disease is seen, but seldomly, to be honest, many of the neurologic presentations don't have that, so if you see a wing-beating tremor, think about Wilson disease, but if you don't see it, don't say this can't be Wilson disease in terms of the neurologic presentations. And then cognitive decline and function, this is typically described as school function in a child, or it could be work function, difficulty balancing a checkbook, difficulty remembering things, any of those things can go along in the adult population, as well as obviously we see people who are in school as adults having decline in school function. Oftentimes they'll say they have trouble thinking in an area, and they'll isolate themselves, so the COD student will find a basement room in some building where there are no people to study, and things like that to try and help them with their cognition. In terms of psychiatric symptoms, again, it's a very broad spectrum, can range from depression and anxiety, oftentimes these people will be diagnosed with adult hyperactivity disorder, attention deficit hyperactivity disorder, bipolar disease, psychosis. Generally though, these Wilson's patients often have what we call a Wilsonian personality, which many of us who see lots of these patients know it when we see it, but I describe it as somewhat disinhibition, it can be associated with decreased executive function, but really the hallmarks are more frontal release, you know, people saying what happens to pop into their brain, or being very creative thinkers, and so forth, is certainly seen. They can be either hyper or hyposexual as well with this disinhibition, and there can be disrupted object relations, you know, with their family and friends and colleagues, particularly at the initiation of diagnosis. So the hepatic presentation, as the previous speaker said, we can have normal liver enzymes, particularly on family screening, abnormal liver function tests, cirrhosis, which can be compensated or decompensated. Obviously we see variceal bleeding in the adult population is something we focus on with variceal banding, and obviously there can be acute liver failure, or acute on chronic liver failure is probably more accurate, since these people have liver disease from the time of conception in the sense that they're not metabolizing copper normally, and oftentimes have a background of cirrhosis when they present with their acute liver failure. Fortunately, that's the minority of the patients who present that way, but we do see those. So in terms of misdiagnoses, I think the previous speaker talked about autoimmune and fatty liver disease overlaps quite nicely. Some of these people will fall in the cryptogenic cirrhosis category. Some of them, obviously if they drink, they get maybe labeled as alcoholic cirrhosis, so any of these liver diseases can coexist with viral hepatitis, and I also point out that PSC and PBC can be confused with Wilson disease with copper accumulation, so if you just look at copper numbers and don't look at histology, you can be misled in that sense. So when we think about therapy in the adults, we think about initial therapy. There can be variable severity at the time of symptoms. As I told you, if we're picking this up on prenatal blood spot testing, the person hopefully doesn't have any symptoms at all at that point, so they can progress to presenting with quite severe neurologic or psychiatric symptoms or hepatic failure, so there's a real variability of symptoms in this initial phase, but we describe this as the time when we start treatment. There's maintenance phase, and I actually like to think of maintenance phase, break it down into maintenance phase, well-controlled, poorly controlled, or over-controlled, because some of these maintenance phase people can actually present as an initial phase in a way if they haven't been taking treatment or haven't been adherent to their therapy. Obviously asymptomatic, and then I'm going to touch on pregnancy as well. Early treatments, as I mentioned, there was BAL, which was an arsenic toxicity biodefense. These were really developed in the UK. Penicillamine, as I said, can be quite toxic, and I'd also like to point out to the pediatric physicians in particular that this is a disfiguring drug, and so one of the concerns I have is obviously the pediatricians don't see that this much, but virtually anyone who's been on penicillamine for a prolonged period of time is going to have progeric skin changes and walk into an adult clinic at age 30 with a neck that looks like they're 80. So this is really a concern I have that the pediatricians may not focus on as much when they say, okay, I'll give you penicillamine because the insurance company is pushing back, but it really is a disfiguring and troubling to someone who walks around looking quite old when they don't have to. Triantine is much less toxic, and obviously zinc acetate or any form of zinc has its own issues with GI upset at times and difficulty with adherence. So these are what we have to work with right now. So triantine really, I think, is taking over as the mainstay when you can access it as initial therapy. The first FDA approval is for triantine dihydrochloride, which is for those with penicillamine toxicity. So you had to make someone sick with penicillamine, and then you could use triantine. We've tried to get around that now. I think most insurance companies in the U.S. are providing it as first-line therapy, recognizing the toxicities of penicillamine, which really forced discontinuation in 25 percent of patients. The data behind triantine, though, the first approval was just a handful of patients. Obviously, I want to credit Dr. Walsh for both these, penicillamine and triantine. But the approval was really on very few patients. They went to the FDA and said, look, these people are dying, and this drug works, and look, we have it in a few patients, and they approved it. There's further data, published data and controls in the ammonium TM experiments, randomized trials that we did at the University of Michigan, and then open-label efficacy studies. And more recently, there's been some very nice work with triantine tetrahydrochloride as well that Carl Heinz Weiss published and his collaborators, and that has been approved as well. So we're having more data that we can fall back on to justify the use of triantine. There's several formulations and manufacturers available. I don't want you to take any commercial bias from this, but I'm just pointing out that you have to be aware that some of these triantine formulations require refrigeration and some don't. The triantine tetrahydrochloride and Reddy's pharmaceutical, and then Cadman, which stopped making the drug, but someone else may pick it up at some point. Their formulations of the dihydrochloride don't require refrigeration, at least not for short periods of time in the case of Cadman, and Reddy's just says you can keep it at room temperature. There's risk of neurologic worsening, so you have to monitor while starting this drug for neurologic worsening, and we generally ramp the dose up slowly and target 10 to 20 milligrams per kilogram as a therapeutic dose. Obviously it depends on what the studies are showing. I'd like to just briefly show that we can treat some people with hepatic failure, decompensated cirrhosis with triantine and zinc, and these people are severely ill. Initial therapy ranging from 1.4 to 2.7, and there are albumins that normalize, prolongations of the INR that normalized, elevations of bilirubin that normalized, ascites in many of these people that normalized, and you can see that they recovered. I also want to point out there's a NASER score and a modified score that are in the guidelines and these can help us figure out who needs transplant and who doesn't. These were developed by pediatricians, so we use them with a little bit of a caveat in the adult population, but we do consult them. So here's a liver biopsy from someone at the time of presentation that was treated with triantine and then zinc, the combination therapy briefly for months and then zinc for many years thereafter, and you can see the scar tissue is really melting down. There's a little bit of fat, but as you can see, looking at the biopsy doesn't jump out. They have Wilson disease, and over time their cirrhosis is really melting away. This is a 10-year biopsy and you can see there's just a very faint band of fibrosis here, and so it's not surprising that a fiber scan says there's mild fibrosis here even though they have cirrhosis. So we can see in long-term biopsy follow-up that the scar tissue is improving with treatment, so I think that's very exciting. Zinc was originally studied as a thesis in the Netherlands in the 1960s and then was developed through work at the University of Michigan. Here I'm just focusing on Dr. Brewer's work here that showed that zinc-induced metallothionein and there was some blockade and a loss in the stool of copper, so the metallothionein copper complexes are shedded off. These are copper-64 uptake studies where you see if someone's treated with zinc, the radioactivity doesn't appear in the blood after ingestion of copper-64, whereas before treatment it does, so this is just showing the blockade. Here I like to point out copper is endogenously secreted through the biliary system over here is what's defective in Wilson disease, and we did copper balance studies, and I really like to show this just to show what happens with dose. So at 25 milligrams once a day, some people are inhibited but not all. At 50 milligrams, we get a little bit more people in negative copper balance, below this line you're in negative copper balance, above you're in positive copper balance, and the goal of zinc is to put you in negative copper balance. As we get up to 50 milligrams twice a day, we start getting more people in negative copper balance, but it's important to point out that if you're taking it once a day, or if you prescribe zinc twice a day and they happen to take one near food, it's not, you don't have a margin for error, and this is why we really like to focus on the 50 milligrams three times a day, or 25 milligrams three times a day, because that gives margin of error for a missed dose, or margin of error, and you can see at these higher doses we're getting everyone into a negative copper balance, but as you can see, there's quite a bit of variability, and some people are barely in a negative copper balance, and some are quite dramatically in a negative copper balance. This goes down to individualization of therapy, and I think the Arhos group is showing this nicely with their copper studies as well, so now with more elegant copper uptake studies it's being shown too. There are two common formulations in the U.S., one is Galzin, the other is Gluzin, they have similar bioavailability, and zinc sulfate is used as well I know in many European countries. The main side effect is GI upset, treatment adherence is an issue, it's generally dosed three times a day, and over-treatment and under-treatment are issues. Obviously there's urine testing, and we monitor 24-hour levels, and I always consider transitioning to Triantin if someone's having trouble with their copper control. Testing is individualized for penicillamine, Triantin, and zinc with assessment of safety parameters, and we adjust based on copper testing. I've mentioned liver transplant, it works, but we really reserve it for the people with fulminant liver disease. I'd like to now just talk a moment about pregnancy. The initial treatment of pregnant Wilson disease patients with zinc was done in 26 pregnancies in 19 women, the health of the mother was well protected on zinc, there were two birth defects, one, a corrected septal defect, and one microcephaly reported in that group. So fairly similar to the general population. Wilson's patients who are clinically stable tend to be able to get pregnant and deliver healthy children, copper reduction treatment needs to continue throughout pregnancy to avoid disease progression. Incidence of birth defects is low, and this has been shown in other studies as well. Penicillamine is a Category D teratogen, Triantin is a Category C teratogen by the FDA. Zinc is generally considered to be theoretically safer, although the numbers of pregnancies are small, and birth defects are so rare that the data is really not robust to make that argument. Ideally, you want to achieve good copper control going into pregnancy. Dose reductions in penicillamine and triantin are recommended. My caveat would be, obviously, assuming the copper is well controlled, you can dose reduce. We have to protect the mother through the pregnancy. And then zinc, I generally let that be guided by the urine copper and zinc levels, and may adjust the dose during pregnancy if the copper levels are very low. Very important to monitor copper control every trimester in pregnancy, and obviously manage varices of presence. So I'll conclude with a summary on the diagnosis of Wilson's disease can be challenging or straightforward depending on the case. The Leipzig criteria and the ASLD guidelines, as the previous speaker summarized, can be very helpful. Presentations of the adult patient can range from asymptomatic to neurologic, psychiatric, hepatic, or a combination of symptoms. Treatment really needs to be individualized based on disease state, medication, tolerance, adherence, and clinical course. And treatment during pregnancy is based on balancing what's best for the mother and the child. Obviously, I'd like to acknowledge the investigators, patients, sponsors, and obviously, I want to reference these treatment guidance and guidelines that have been put out. So thank you very much for your attention. Thank you, Dr. Ascari. Our next and final speaker will be Dr. Michael Shilsky from Yale University, who'll be talking about emerging treatments and current trials. So thank you to the entire Pediatric SIG for having this whole session on Wilson's disease. This is wonderful. And to our organizers, Nanda and Jamie, for really getting a wonderful panel of speakers today. It makes my job easy, because the table is really well set. Do we have to use the... All right. So we do owe a tribute to Wilson himself. Even though he trained in the UK, he was actually born in the United States, in New Jersey. So we have a partial claim to him. He was the one who really put this on the map for liver disease in the sense that he recognized at the time of necropsy of his patients that it was important to put the hand under the diaphragm, and you can actually feel the cirrhotic livers of the patients that he had that succumbed to the disease. So we do owe him a debt of gratitude for putting his monograph forth and really stimulating the thought that there was a toxin, which we now know as copper, that caused the disease. And for those of you who recently felt a vibration and a shake in the force, we're very saddened at the loss of John Walsh, who contributed so much to the field of Wilson disease. He did survive two years past getting his letter from... So, as Fred had mentioned, the timeline for development of drugs, it was a 50-year period from Wilson's wonderful monograph until the first oral therapies came out. And now we're in the era where we've, and I'm going to share with you some updated data on the Triantin, because that really is, even though I'm talking about emerging therapies, we're going to talk about emerging monitoring that goes along with it. And then a little bit about where we are in the development for tetraethylamyelithate and the exciting era now of gene therapy for Wilson's disease. So this is taken from our new guidance. For those who have been frustrated looking for it on the website, it does exist in a pre-proof form out there, but it doesn't appear on the guidance page or guidelines page yet. That's because the proofs are being fixed. For anybody who's ever written with Eve Roberts knows that you have to have every single I and T perfect. So we are going to get it perfect, and we hope within another week or two, our copy editors won't keep causing us grief and we can get it out there for you. But when we think of treatment, we have to think, and especially in a disease where you have such diversity, there still is a natural history, it's just that the timeline differs for many individuals. So if we look at this as an untreated natural history, timeline starting on the left, moving to the right, ultimately leading to death, there are different phases of the disease. And when we think of treatment, we have to recognize that the treatment may be tailored to the phase of disease. In the early asymptomatic phase, you want to prevent disease progression. When you have symptoms or tissue damage, you want to treat that and try to bring them backwards towards that preventative phase. And then when you have those that are well advanced and complicated, you have to go into rescue mode, the kind of therapy that Fred had talked about in terms of combination therapy, or there may be other rescue attempts by other antioxidants or other measures to try to reduce the copper. But again, each phase of the disease evokes a different response from us in what our treatment should be. Now, this is sort of a summary. It does not appear in the guidance. It's something I made after in my presentation to the apposal group when they asked us to sort of correlate the information. But what it amounts to is, first, always verify the diagnosis. And then again, as I mentioned, determine the state of the disease. And then when you can put them in that asymptomatic bucket, you're going to assess them for tissue damage. And if they actually have tissue damage, we take those patients and we move them out of the asymptomatic bucket and deal with them similarly to the symptomatic patient. And then those individuals get a higher dose of chelation therapy. And I will credit our pediatric colleagues, audience here, Eve in particular, who've actually made our adult hepatologists think in milligram per kilogram dosing, because that is really something. It was a one-size-fits-all for adults before. And I think it's important to really recognize that we have to start standardizing our dosing and treatment for patients. And with the standard currently available triantine and penicillamine compounds, we typically treat the symptomatic or tissue-damaged patients with 15 to 20 milligrams per kilogram with the aim of stabilizing them and returning them back to maintenance therapy, which would be the same way we would start for an asymptomatic patient as well. All right, so let me turn to the study that Fred was alluding to, and this was the first study comparing the tetrahydrochloric compound of triantine versus penicillamine for maintenance therapy. So even though this drug has been around for a while, there really had not been a proper testing of it in the maintenance phase with data available. And this was a prospective open-label trial. And the open-label trial led us to actually have to think differently, because you then have to then come upon an objective parameter for measuring what your outcome is. And this, indeed, our primary endpoint was the non-ceruloplasmic copper. That prevented bias from coming in from the observers for the individuals as they were randomized into the different arms. So what was developed is a new method, and this is not yet commercially available. But we have been using it in research study in collaboration with the group in the Royal Surrey Hospital. And then this is the group that had been doing the work that was in the published document in the Keylight study itself. And what it involves is taking the serum sample, separating the serum proteins using anion exchange chromatography, so that you can isolate the ceruloplasmic peak from the other protein peaks. And then you can determine the copper uniquely in the ceruloplasmic peak. This methodology has about 98 to 99 percent recovery of the copper. And so, therefore, it's really a robust, a precise, and reproducible methodology that's really very helpful. In the study, which was a comparison of the triantin-randomized patients to those that stayed on penicillamine, what you can see is this is non-ceruloplasmic copper on this axis, and this is to the right is study week on the x-axis. There really is a very tight range throughout the study for both, and actually a little bit of study effect that the values get lower in both groups over time. That's because patients probably adhere to their diet better, taking their medications better in a clinical trial. But it shows you how robust it is in NCC. Now, the expectation in this trial, starting with stable patients, that you're going to end with stable patients, and what you see in the end is a non-inferiority of the tetrahydrochloride to the penicillamine, but what it introduced as this primary endpoint really was the ability to look at this assay. And what we believe happens, and this needs to be validated further, we've seen this in experimental models when people looked at exchangeable copper, which is another way of looking at bioavailable copper, that the elevation in that bioavailable pool precedes the development of tissue damage and clinical disease. So it's important to be able to get at that pool. So the secondary thing that we learned from the study is that this is the penicillamine arm, and below you see the triantin arm, and you can see less urine copper excretion for comparable doses in milligram quantities of the drugs. And it's fascinating, because the NCCs stayed the same in the two arms. So what this means is that there's probably a secondary action of the triantin, not just to cause urinary copper excretion, but we postulate probably to block copper absorption from the gut as well. And if you indeed look at the pharmacokinetics, penicillamine is almost 100 percent absorbed. Triantin is not. It's actually a much lower percent absorption. So we think that there probably is two actions of the triantin compound, and that explains the difference in this. So the lessons. So again, primary endpoint, using a very objective, new, accurate measure of non-seruloplasmin copper. So as Fred said, you know, say your goodbyes, and put the nail in the coffin on the old estimated value for NCC. And we think that this really is going to be important for the future. And again, as I mentioned, mechanism of action becomes interesting if we start looking at objective data, and that'll have to be looked at more critically as well, using other methodology. But it will be important. This was a stable population of patients. So we yet have to show that the NCC, how it behaves in an unstable, initially untreated population, and then follow that over time, and that's sort of our next phase of work. So we're going to move on to some of the experimental treatments yet. The choline tetraethymalidate, which I'll show you in a second. And then the AAV-mediated gene therapy, of which studies are initiated as well. So this is the molecule tetraethymalidate. It's really a fascinating compound. And the addition of the choline form of the molecule allows stability to occur. In the original studies with the ammonium form, it was a very unstable molecule, oxidized quickly, needed to be stored under argon, and we used to have to get a chip from the University of Michigan every month as a supply, and our pharmacists kept it tight under control because of that instability. But the choline form actually is a very stable form in terms of its oxidation, and what it does when it's released into serum, it creates a complex, as you can see on the right, with a fairly high binding that has copper in it, along with the molybdate bound through the sulfur groups, but it actually creates a complex with albumin in the circulation that's very tight. And that creates different issues in terms of monitoring as well. One more thing about its properties, and shown in the little orange bars are the binding for the KDs for both penicillamine and triantin, PA and TR, compared to the tetraethymalidate. You can see a couple of order of magnitude different. So it's a very, very tight binding with tetraethymalidate to copper. Tetraethymalidate is not promiscuous in that it's not binding other metals, whereas some of the other chelators do have the ability to bind other metals as well. Shown on the right here, I'm not going to, it's a little hard to see, but very simply what it shows is that tetraethymalidate is one of the few chelators that has a high enough binding constant to actually pull copper out of metallothionein, a storage molecule. And that is really an interesting property, in theory can get to different pools of the copper that may not be available to the standard chelation. So the phase three study had been completed, we're waiting for the final data, and Fred is going to give us on Sunday a summary of the phase three study in a separate session. There were two cohorts, there were those that were naïve or early to treatment and those that were previously treated. It was open label, it was randomized whether they would stay on their standard of care or to the tetraethymalidate, and we'll talk a little bit about the unique assay in a moment, but all these patients were monitored biochemically and then clinically and then also for safety during the study. And the primary endpoint shifted during the study and it became the area under the curve of the labile copper. And so what is that? That's another way to get at that bioavailable copper. This is the assay, it will be presented as a poster as well on the 6th of November. And what had to be done here was a way to get at, to take out this real plasmid copper and then isolate and look at separate the copper that is in that tetraethymalidate copper albumin complex, because that's inert and non-reactive and you need to know what was left in terms of that labile copper. So this was accomplished through an immunoprecipitation assay using ceruloplasmin antibodies to pull out the ceruloplasmin. And then secondarily, there was sort of a similar method to the exchangeable copper in that EDTA was used to pull out copper that was not bound tightly to the albumin complex and that's left what's called the labile copper. I won't belabor that too much, but just to tell you that just look at all the three pools in the cohorts of the study. On the left axis is the mean copper concentration appearing in the blood, and over time is the weeks. And on the left side, you can see this is the cohort that had been treated for a long period of time before. On the right side is the cohort that's the either initial treatment or relatively short period of treatment. And what I just want to point out is that each of these, even in a well-treated population, with the addition of tetraethylamylipidate, copper is coming out into the circulation. Okay? So that means there is more bioavailable copper and there is a potential to pull copper out from certain pools in the body, and that occurs obviously more so to a greater magnitude in the untreated or early treated patients. But the exciting part to me was that this is interesting because we always think of our patients have been treated long term with our other standard of care therapies or any way to remove copper from other sites. Now the question will be, what's the disposition and are we seeing an improvement in these patients? And I'll leave that to some of the presentations later on. But to me, just the fact that these are mobilized is a very interesting thing that we've actually been able to show now. Okay. So on to the last thing here. And you can see all the way on the right, which is the vector of choice that has been found for two studies that have been launched, which many ATP7B genes have been inserted into the adeno-associated virus. These are small, non-integrating, replicative defective viruses. And you can use them sort of as a Trojan horse for introducing genes. The right serotypes have great ability to direct themselves, target direct into liver cells where they're highly taken up. And that great creates an advantage in that whatever you're going to give is really going to be directed to the liver. And you'll see other gene therapy studies for neurologic disease and other where there is off-target problems because the vector may not be as well suited to get into the particular tissue type as AAV is for liver. There's a nice review in emerging issues in AAV-mediated in vivo gene therapy. And it appeared in one of the cell press. And it sort of highlights the fact that this goes into the cell. And ultimately what you're aiming for is an episomal AAV genome. And that integration events are extremely rare, unlike viruses like lentivirus where you find that it's meant to integrate in the cell. Now the downside to that is, again, you have to keep that cell alive to be able to then have the episomal AAV because it's not necessarily passed on to the next generation of that hepatocyte. The other thing that happens is you can get the coat, then present it to the body again by our antigen presenting systems. You can develop neutralizing anti-capsid antibodies. And in fact, in our population of patients, there's always, all of us, a percent of us have been exposed to the AAV in nature itself. And we then have to find people for these studies who don't have the neutralizing capsid antibodies present to be able to start with that. And then obviously the other part is that by doing the therapy, what we need to do is find ways to prevent this part of the pathway from occurring as robustly so that we may have the opportunity to retreat some of these patients. There was some wonderful preclinical work done by two separate groups. And this was published in Hepatology and the other in Human Gene Therapy Clinical Development. And what these both show is that the mini-ATP7B gene, and you can't take the whole gene because the AAV doesn't have that big a stuffer region that you can put things in, so you have to miniaturize it and still have a very functional gene. And this has led the way to, through a lot of other work that we don't have time to talk about, to allow for the clinical trials to have started. And there are two trials, one using, both using the different mini-ATP7B genes that are very similar in function. And there's one that is conducted by Vivet and another by Ultragenyx. And there are many different sites that have been recruited in Europe and in the United States to start. And both of these are a single-dose introduction of the gene and then looking for outcome from that. There's a wonderful follow-up study in molecular therapy which actually talks to mechanism of action of the gene therapy. It's very important to reestablish that copper excretion, preferably by standardized pathways. And if you introduce the ATP7B gene and then you follow up the copper, if you give it exogenously and then you look for the uptake in the parallel animal model of Wilson disease, you'll see that it sticks in the liver, doesn't go anywhere else. And the reason is it's mixing with a high copper pool and not getting out into the bile. If you introduce the gene back in, you can start to see development at 24 hours of copper that's getting out in the bile into the intestines and at 48 hours, a diminution of the presence of what was in the liver itself further going into the gut. And the parallel part is the incorporation of copper into ceruloplasmin and reappearance back in the blood as well. So these kind of mechanism of action studies need to be coupled with the therapeutic trial so that we will actually learn about the efficacy separate from the safety for the trials. So the end points, ultimately we hope that you're going to permit the safe discontinuation of standard of care treatment and you may ask, why do we want to develop these kind of things for patients when we had all those other wonderful drugs? And the answer is 50% of our patients are non-adherent during the course of a lifetime, some of them as we know with devastating consequences. And when you ask patients themselves, they say, I want to be able to eat all the things that everybody else does, the chocolate, mushrooms, all the goodies. And so ultimately we want to show functional expression and then maintenance of stability in these patients, both biochemical and clinical. And then we have to show that in a manner that it is safe as well. So questions, how safe is it? How long will it last? Can you repeat the treatments? And just obviously we hope it will never preclude an alternative treatment or retreatment in the future. These are all questions that are going to be answered in the very near future. And what about other options, emerging things for the future? People are thinking of integrated viruses. Again, there's safety issues involved with that. And so they'll have to make sure their targets are 100% without off-target insertions. There may be opportunities for gene repair, although as we know there's a huge number of mutations out there. So there have been recently some discussions of large segment CRISPR replacements. And if that comes to actual fruition and is accurate, that may be a little bit of a game changer in terms of the economics of trying to go after particular areas of a gene as opposed to doing a single mutation. There have been interesting work about people who have been looked at. Some of the variants of the ATP7B protein resulting from mutations that are indeed partially functional and that there may be the ability to use chemical compounds to sort of as chaperones to move them into functional parts of cells. Cell replacement has been an issue because using hepatocytes has generated problems in terms of immune suppression. However, if the use of stem cells, interstitial cells that can be reprogrammed into hepatocytes from individual patients is actually shown to be robust in the future, that may be fascinating. There are also other targets within the cell. People have been looking at whether the unique copper transporters that may be particular CTR types on individual cells may be important and we might be able to regulate them. And the other off-target things that may ameliorate cell injury independent of the copper work by Jamie Hamilton and the group at Hopkins has looked at the FXR receptor agonists and has proposed that may ameliorate injury as well. So in summary, we're still learning a lot more about our current therapies and certainly emerging treatment monitoring is going to be on the horizon. Experimental treatments for Wilson disease do address some of our unmet needs for patients and these curative therapies are still in development. And I'd like to acknowledge you all for organizing this session. The recognition that we need academic industry partnership to accelerate the development timeline for these therapeutics and monitoring is really, I think, is just very practical and very honest these days. I'd like to thank the Wilson Disease Association for their support for patients and their mission for supporting research and the many investigators and staff that made these possible. So thank you very much. Thank you so much, Dr. Shilsky, and I'd like to invite the other speakers up to the podium so we can start our question session, question and answer. If you'd like to come to the mic and introduce yourself and then ask your question. And I'd like to start that off actually to Dr. Shilsky, who's spoken a lot about the non-sirloplasmin-bound copper and the assay for it, and there's been a lot of research going on about it for several years. And I wonder how long it'll take to come to the market and if people do want to get it done, is it available on a research basis? The answer is we hope sooner rather than later, but of course, you know, when things are being done for clinical purposes, you have to have clear certification. And so there is some efforts in that direction. The pandemic really created a lot of problems because any laboratory that wanted to be clear certified couldn't be visited during that time. Those restrictions have been lifted, so hopefully that will be accelerated in terms of timeline. Unfortunately, outside of clinical trials that are not really available, there will be a phase four trial being launched for the tetrahydrochloride compound, and in that will be the aim of a lot of validation around the assay and utility of that. Okay, thank you. Please. This is Samar Ibrahim from Mayo Clinic. Thank you for really interesting talks. My question is for Dr. Kamath, is about overtreatment in the pediatric population. This is really a challenging issue, especially, as you mentioned, if we calculate the non-sirloplasmin-bound copper is negative, and I know there are new methods available, but it's not for clinical use yet. So, thanks, Samar, for the question, so, which is how do we assess if it is overtreatment in that setting? I mean, I think it's hard, right? So if you can't rely on your non-sirloplasmin-bound copper, you could, for a start, you could potentially ask your lab if they can get an assay that doesn't overestimate the herlis sirloplasmin, because it's not all assays that do, so that's one thing that you could do. You can look at years of treatment. You can look at extent of organ damage, because, you know, we can use all our other clinical parameters that we have to see, you know, have we assessed this child, have we, how much copper have, or damage have we minimized in this child, and then try and assess it from there. And I think the safest thing always to do in that setting, I would say, is if you stop chelation and continue with zinc, and then see what happens. And if the child's liver parameters stay stable, then probably it was a situation, but if they start to rise again, then maybe that wasn't the situation. I think it's tricky if we don't have an assay. Do you have a better answer? Well, I think most of the overtreatment is seen in the adult population. You're seeing them for 20 years approximately. We see them for the next 50 to 60. And so it carries over into our cohort. We do see a perturbation in iron. You lose further ferroxidase activity, so you may see higher ferritins. You may see on a biopsy tissue accumulation of iron as well. So you can get this sideroblastic anemia and then tissue iron deposition as one evidence. The sort of canary in the coal mine is often the neutropenia, though. And this is independent of direct drug toxicity. And the other thing we see in the adult population is the shift in the neurologic exam when you start to see demyelinating problems akin to what had been reported to the zinc-containing dental compounds and the zinc toxicity that had occurred in the over-ingestion in those individuals. So you may see demyelination that can be evident on MR scans as well. Thank you. Roy Coley from Los Angeles. Very nice group of presentations. Thank you for the organizers. Quick question to Dr. Shilsky. The last slide, the last few words, you mentioned FXR. So I had to get up and ask a question. Are these FGF19-driven pathways or are these FGF19-independent FXR effects? I'll leave you to ask that of Jamie, directly Jamie Hamilton. Okay. Hi, Richard Thompson from King's. A great session. I enjoyed all the talks. I've got several questions, but I'll restrict myself to one to Dr. Medici, actually, which is about the HDAC observations you made at the beginning in the methylation. And I wonder about the zinc status of the patients when those samples were taken. I wonder if any of them were zinc deficient because HDACs are very dependent on zinc for their function. So, no, actually, I didn't say it, but these patients that we studied on HDAC, these were pre-treatment. Exactly. So at the time of diagnosis. Which pre-treatment, many patients are zinc deficient, aren't they? I don't know if I would say if we know of an actual zinc deficiency in patients with Wilson disease. I didn't have that evidence, but yeah. So there is interesting data. I think maybe Professor Thompson was referring to, from Anil Dhawan, where he's noticed low zinc levels in the acute liver failure patients, and that's being revisited. Okay, we'll have one time for one last question. Hi, it's Aftab out of London. You mentioned Kaiser Flusher rings. Everyone talks about Kaiser Flusher rings. But quite a qualitative exercise. So I wonder whether you've got experience of other modalities you see as the future for Kaiser Flusher ring evaluation, and whether you feel the current guidelines put too much emphasis on that. In fact, there's a point of two when, in fact, if the KF ring is probably there but not picked up by an adequate ophthalmologist, it could maybe falsify the diagnosis. So I wonder whether the group had comments on that. Well, there are efforts to quantify KF rings, as you know, by optical adherence tomography, and that may well be a way in the future. KF rings are always a subjective finding, as you're pointing out. This is really someone's opinion about whether they're there or not. People who have light blue eyes tend to have the KF rings observed more than people with dark brown eyes, for example. They're just harder to see. And at what point you say there's a subtle ring present and you're giving it two points, you know, is a thing. You know, the point scoring systems are wonderful, and they're good to get us thinking about everything, but, you know, I'd like to emphasize that just because someone has four points on a Leipzig scoring system doesn't mean they have Wilson disease. It has to fit clinically with everything else that we see, because there are ways to get four points without having Wilson disease. So I think it's a good point that you make, Avtav. Okay, well, thank you all very much for attending this session and making it successful. Thank you. Thank you all.

Wilson disease

genetic disorder

ATP7B gene

copper accumulation

liver damage

neurological symptoms

psychiatric symptoms

copper metabolism

oxidative stress

DNA damage

diagnosis

treatment options

ongoing research