Please welcome to the main stage, Dr. Jad Spheer. Good morning. Welcome everyone to the Bone Plenary Session. Before we start, I'd like to remind everyone to silence your cell phones. We will have time to address questions at the end of the session at the microphones between the audience. Now it's my pleasure to introduce our Bone Plenary Speaker, Dr. Raj Kumar. Dr. Kumar is the Ruth and Vernon Taylor Professor of Medicine and Biochemistry and Molecular Biology at Mayo Clinic. He has practiced nephrology and internal medicine at the Mayo Clinic for the past 41 years. He has dual appointment in the Division of Nephrology and Hypertension, as well as endocrinology metabolism and diabetes. He has research interests in vitamin D metabolism, calcium and phosphate homeostasis, and in the mechanisms of bone loss mediated by tumor. This morning, Dr. Kumar will talk to us about phosphatonins from discovery to therapeutics. Please join me in welcoming Dr. Kumar to the stage. Good morning. Thank you for coming. I'd like to thank the organizers for the opportunity to speak here at this meeting. And I'd also like to thank the introducer for that introduction. It's the kind of introduction that my father would be proud about, but only my mother would believe. So today we're going to talk about the phosphatonins, and these are hormones that are involved in phosphate transport. And this is an example of the application of investigation to a clinical problem that has resulted in new paradigms. And we've been successful in doing so because of close collaborations with individuals within Mayo Clinic, in particular Steve Hodson, Peter Tebben, and Bart Clark, and also individuals outside of Mayo. And in particular, I'd like to recognize Dr. Jandipur from Johns Hopkins University, Susan Schiavi from Genzyme, and Mark Dresner from the University of Wisconsin, with whom we've had close collaborations over the years. Now in the outline of the talk, I'll talk about phosphate homeostasis in mammals. I'll talk about the hormones involved in phosphate homeostasis. And I'll talk about various factors that are generated by organs other than bone that may regulate phosphate transport, in particular the intestinal phosphatonins. And then finally, I'll talk about an approach to the diagnosis and treatment of hypophosphatemic disorders, focusing on tumor-induced osteomalacia, with a few comments about XLH and the use of FGF23 antibody that is directed against FGF23, and is helpful in the treatment of these disorders. Now phosphorus plays an important role in cellular signaling and the maintenance of membrane structure, cellular energy and intermediary metabolism, nucleic acid metabolism, and of course, something that you're familiar with here in this audience, bone mineralization. So it's easy to infer that changes in phosphorus concentrations will have important consequences with respect to the wellness of the organism. Now low phosphorus concentrations, especially when they're very low, are associated with rhabdomyolysis, decreases in cardiac function, and altered neutrophil function. And obviously in the case of nutritional osteomalacia, one can get changes in bone mineralization. Now shown here on this slide are the processes that are involved in mineralization of bone. Shown on the left of the slide is a bone mineralization unit with osteoclast resorbing bone and osteoblast coming behind and laying down bone matrix, which then gets mineralized to give rise to hydroxyapatite and mineralized bone. There's a series of reactions that occur that allow mineralization to proceed, and those are shown on the right side of the slide. Now shown in this slide is an example of what bone would look like in a person with severe phosphate deficiency and osteomalacia. In the top panel on the left is normal bone stained with a goldness stain, and you can see that mineralized bone is a greenish blue hue, and unmineralized matrix is orange. Shown in the lower left is mineralization rate that is determined by the administration of tetracycline fluorocombs, and by measuring the distance between the two labels, one can get an idea of how efficiently bone is being mineralized. On the right hand side of the slide is shown what happens in osteomalacia. There there's an excessive amount of mineral, unmineralized matrix, which is unmineralized because of a deficiency in phosphorus, and shown on the bottom right is the fluorescent label pattern that is seen in such a case, where there is very little uptake of double-labeled uptake, and the difference and the separation between the two labels is very, very small. So, the mineralization rate in the case of osteomalacia is greatly disturbed. Now, there's also good evidence that comes from individuals with chronic renal disease, showing that high concentrations of phosphorus are associated with cardiovascular disease and excess mortality, and this is a summary of a number of studies that have been done looking at mortality and cardiovascular morbidity in patients who have hyperphosphatemia. And on this slide you can see that as chronic renal failure progresses and GFR drops, phosphorus levels increase, and this is associated with an increase in mortality and morbidity. So I think it's clear that reductions in phosphorus and increases in phosphorus are associated with bone disease. Now this is a slide that shows that if you reduce serum phosphorus concentrations, you can reduce cardiovascular mortality or morbidity, and these are studies done with the use of sevelomer, and you can see that decreasing phosphorus concentrations are associated with less morbidity and cardiovascular mortality. Now in this busy slide are shown some of the numbers that are relevant to phosphorus metabolism, phosphate homeostasis in normal humans. There are three organs that play an important or a crucial role in the maintenance of phosphorus concentrations, and these include the intestine that is responsible for the absorption of calcium, of phosphorus, excuse me, and the kidney that is responsible for the excretion of phosphorus. In the extracellular fluid, phosphorus concentrations equilibrate between ECF and also bone, and one can get shifts of phosphorus from the ECF into tissues that can alter phosphorus concentrations. But in states of phosphorus balance, the amount of phosphorus that is being absorbed by the intestine is equivalent to the amount of phosphorus that is excreted by the kidney, and the numbers should be about equal in states of balance. One can measure the efficiency of phosphorus absorption in the kidney by measuring the fractional accretion of phosphorus or the tubular maximum for phosphorus and can infer whether there is a loss of phosphorus that occurs as a result of renal mechanisms. Now this slide shows the current understanding of hormones that are involved in phosphorus homeostasis. PTH drives down serum phosphorus concentrations and induces a state of negative phosphate balance, whereas vitamin D3 increases the absorption of phosphate both in the intestine as well as in the kidney and induces a state of positive phosphorus balance. But I think the regulation of phosphorus is really much more complex than is currently thought about. There are both short-term and long-term regulators of phosphorus concentrations of phosphorus homeostasis. Long-term regulators, shown on the bottom of the panel on the left, include things like PTH, the phosphotonins, FGF23, SFRP4, FGF7, and FGF2. The factors that increase the retention of phosphorus still include vitamin D3. The concentrations of vitamin D3 are regulated by long-term regulators as well as short-term regulators. The short-term regulators are those that are responsible for the alteration of phosphorus concentrations or the maintenance of phosphorus concentrations. On the short-term, that is within a period of 10 to 15 minutes after absorption of a meal or taking a meal that is high in phosphorus. This slide shows the mechanisms that come into play when a normal serum phosphorus concentration is perturbed. So shown in the orange are changes that occur once serum phosphorus concentrations increase. The increase in serum phosphorus concentrations is associated with a decrease in serum calcium concentrations, an increase in PTH that changes the efficiency with which phosphorus is absorbed in the proximal tubule. This tends to bring phosphorus back into the normal range and restore phosphorus balance. On the other hand, when serum phosphorus concentrations are decreased, there is an increase in the synthesis of 125-dihydroxyvitamin D3 and there is an increase in intestinal and renal phosphorus absorption, all of which tends to bring phosphorus concentrations back towards normal. Shown in the blue on the right-hand side of the slide are changes in serum calcium that will occur when phosphorus concentrations are reduced, showing that serum PTH tends to go down in the presence of hypophosphatemia and causes an increase in renal phosphate retention. Now, it turns out that there are a number of growth factors that regulate the efficiency of 125-dihydroxyvitamin D3 synthesis. These factors include IGF-1 that goes up when serum phosphorus concentrations are decreased and FGF-23, or fibroblast growth factor 23, which I'll talk a little bit more about, which tends to go up when serum phosphorus concentrations are increased. So the interplay between short-term factors, as shown on the right of the slide, and more long-term factors, as shown on the left of the slide, and the interaction with growth factors is important in maintaining normal phosphorus homeostasis. Now there are several clinical conditions that can cause hypophosphatemia, and we will go through in a slide that I'll show subsequently an approach to diagnosing which one of these might be present, quite apart from the history and by the use of various laboratory methods. One can get an increase in, a decrease in dietary phosphorus intake as a cause of hypophosphatemia. One can have vitamin D deficiency or vitamin D resistance that causes phosphate deficiency and a malabsorption of phosphate, and the use of excessive amounts of phosphate binders can also result in hypophosphatemia. I think in many cases there is a shift of phosphorus from the ECF into cells, and respiratory alkalosis is very frequently responsible for hypophosphatemia that occurs in patients who you might see in the hospital. A number of hormones such as insulin, epinephrine, and cortisol can also change the amount of phosphorus in the ECF. Nutrients such as glucose, lactate, and amino acids can do the same, and one can get changes in the amount of phosphorus being taken up by tissues in clinical states such as recovery from hypothermia and following parathyroidectomy. But I think by far and away the commonest cause of phosphate changes or decreases are losses that occur in the kidney as a result of hyperparathyroidism, as a result of oncogenic osteomalacia, and as a result of inherited rickets that I'll talk a little bit more about as we go on. Now what are the phosphotonins? The term was introduced to describe a factor or a number of factors that are responsible for changes in renal phosphate reabsorption seen in patients with tumor-induced osteomalacia. These patients typically have hypophosphatemia, hyperphosphateria, and a low TMP by GFR, normal 25-hydroxyvitamin D concentrations, and low 125-dihydroxyvitamin D concentrations that contribute to hypophosphatemia by reducing the amount of phosphate absorbed in the intestine and also in the proximal tubule of the kidney. These individuals generally have low normal or low normal 125-dihydroxyvitamin D concentrations and they have normal PTH concentrations. A bone biopsy will show a mineralization defect and the presence of osteomalacia, and this is typically manifest as bone pain that the patients will present with. So all these biochemical abnormalities disappear when the offending tumor that is responsible for some of these biochemical defects is removed. Now we had the opportunity to study such a patient and take tumor cells and put them in culture, and what we showed was that if you took supernatants from tumor cells, there was no change in alanine or glucose transport, but there was a profound inhibition, about 50%, in phosphate transport. And we also found that there was predominantly a change in TMP by GFR and a change in vivo in the amount of phosphorus that was being lost. We also found that unlike PTH, which caused an inhibition of phosphorus transport as seen on the far left, in association with an increase in cyclic AMP, tumor supernatants caused an inhibition of phosphate transport without changing cyclic AMP. So this told us that we were dealing with something that signaled in a way that was different than PTH, and PTH was not responsible for many of the findings that we observed in the patients. Now, we found that implantation of tumor cells in nude mice recapitulated the phenotypic features of the syndrome. Namely, there was hypophosphatemia, a bone mineralization defect, and low 125-dihydroxyvitamin D concentrations. So in an editorial that accompanied our paper, Econs and Dresdner thought that we had unveiled a new series of hormones that were responsible for changing phosphate transport in the kidney. And just to summarize, these substances cause phosphaturia, they inhibit 1-alpha-hydroxylase activity, and possibly interfere with mineralization directly rather than through a change in phosphate concentrations. Now, the next important advance was made by the ADHR Consortium, who was studying a group of patients with autosomal-dominant hypophosphatemic rickets. These individuals have manifestations that are more or less similar to those seen in patients with tumor-induced osteobalacia. And they noted, using positional cloning experiments, that there was a mutation of a new gene called FGF23 that was responsible for many of the phenotypic features found in these individuals. Now, we did subtraction mRNA sequencing to determine what factors were upregulated in tumors of individuals with osteomalacia. And we found that a number of proteins, including MEPI, SFRP4, and FGF23, were upregulated in the tumors compared to tissue that was normal. So I think there's good evidence that many of the phenotypic features observed in these patients are due to upregulation of the proteins shown in orange, namely MEPI, SFRP4, and most importantly, FGF23. Additional candidate phosphotonins include fibroblast growth factor 7 and FGF2. But I think the data that support the use or the role of these factors is less compelling than it is with the previous three proteins that I talked about. So let me show you some data that demonstrates that FGF23 and SFRP4 inhibit phosphate transport both in vivo and in cultured cells in vitro. On the bottom panel, you can see the effects of increasing amounts of FGF23 and SFRP4 on TMP by GFR, which gets progressively inhibited as you administer more and more of the peptide. Likewise, in the top panel, you can see that the uptake of phosphate in proximal tubular cells is reduced as you add increasing amounts of FGF23 or SFRP4. Now shown here is data that MEPI, which is a matrix phosphoprotein, also increases phosphate losses from the kidney and also decreases the fractional excretion of phosphate. This is data from Rowe and his colleagues now at the University of Kansas. So both FGF23 and SFRP4 reduce phosphate reabsorption, and they do so by reducing the number of sodium phosphate co-transporters that are expressed on renal tubular epithelial cells. Now just to take you back to physiology, medical school physiology, a bulk of phosphorus is reabsorbed in the proximal tubule by a series of transporters, the main one of which is called sodium-dependent phosphate transporter 2A or NAPI 2A. And one would infer that the substances that cause phosphaturia would change the number of transporters that are present on the cell. So here's pictures of some proximal tubular cells that are expressing fluorescently labeled sodium phosphate co-transporter. And in the top left you can see on the vehicle that the numbers of transporters on the surface of the cell, shown in green, are fairly abundant. You can also see on the bottom right that as you add FGF23, the number of transporters disappears. And the same sort of phenomenon is observed with PTH1-34. And the same kind of findings are recapitulated with SFRP4. So I think it's clear from this experiment that the changes in phosphate transport that are observed in vivo are due to changes in the number of transporters present on the surface of the cell. And a reduction in the number of transporters by FGF23, SFRP4, or PTH causes a change in the efficiency of phosphate uptake in cells. Now it turns out that FGF23 is biologically active only after it binds with a protein called clotho. And clotho forms a heterodimer with FGF23 that allows intracellular signaling to occur. And one would infer from this that changes in clotho may indeed be associated with changes in phosphate. And this is true in certain sporadic cases of hypophosphatemia in which clotho levels are altered. And also in the case of chronic renal failure where the amount of clotho that is present may be reduced. The SFRPs have a much more complex mechanism of action. You can see on the right side in panel A that under normal circumstances Wnt proteins bind with a frizzled receptor after associating with a co-receptor called LRP56. And when there are excessive amounts of secreted frizzled related protein as shown in B, signaling into the cell using the canonical pathway no longer occurs. And so the reason SFRP4s cause hypophosphatemia is that they interfere with Wnt signaling in the tubule. Now another way in which the phosphotonins reduce phosphate transport is by reducing 125-dihydroxyvitamin D3 production by inhibiting the 25-hydroxy-D-1-alpha-hydroxylase or the CYP27B1 enzyme. And this slide taken from Shimada and his group shows that if you take CHO cells that overexpress FGF23 and then look at the numbers of molecules of the 1-alpha-hydroxylase that is present, it is reduced compared to cells that have been transfected with a vector that does not express FGF23 or with PBS. So it's clear from in vitro experiments that there is a reduction in 1-alpha-hydroxylase activity. The same group went on to show that if you knocked out FGF23 gene expression using knockout animals, that there was an overexpression of the 1-alpha-hydroxylase. And this occurs quite early following the knockout of the FGF23 gene. So at 10 days in the top panel on the left, you can see an increase in the amount of mRNA for the CYP27B1, and it persists at 3 weeks and at 8 weeks following the knockout. And this is associated, as you can see in the bottom panel, with a reduction in the amount of protein, the CYP27B1 protein, that one can detect by immunohistochemistry. This, of course, is associated with hypercalcemia in the animals at 10 days, 2 weeks, 6 weeks, and at 9 weeks. And this is consistent with the observation that patients who have tumor-induced osteomalacia have low 125-dihydroxyvitamin D3 concentrations. This slide demonstrates that SFRP4 does the same sort of thing, namely inhibits 1-alpha-hydroxylase activity. And if you transgenically overexpress SFRP4 in mice, there is a reduction in 125-dihydroxyvitamin D. Now, there is a feed-forward loop such that if you administer 125-dihydroxyvitamin D3 to an animal, there is an increase in FGF23. So that increases in phosphate that are brought about by 125-dihydroxyvitamin D3 are regulated by an increase in FGF23. And this is shown as a cartoon here. A reduction in phosphorus results in an increase in 125. This causes an increase in serum phosphorus concentrations and an increase in FGF23. And FGF23 negatively feeds back on 125-dihydroxyvitamin D3 production, as does an increase in phosphate. So there is a feedback loop involving FGF23 and 125-dihydroxyvitamin D that is physiologically relevant. Now, there are a number of conditions which are associated with increases in phosphotonins or phosphotonin activity. These obviously include TIO that I've spent some time discussing, X-linked hypophosphatemia, autosomal dominant hypophosphatemic rickets, autosomal recessive hypophosphatemic rickets, and renal failure. And in each one of these conditions, there are changes in FGF23, SFRP4, and FGF23. And I'll discuss these in a little more detail here in the next few minutes. So here is data from Johnson and others showing that if you remove the tumor that is associated with the generation of phosphotonins, FGF23 concentrations that are initially quite elevated, as shown on the left, come down after surgery. There are also increases in FGF23 concentrations in people who have X-linked hypophosphatemic rickets. And together with Mark Dresner, we overexpressed FEX, which is the mutated enzyme, in XLH, in osteoblasts, or in all cells of the body. And one noted that when there is a global knockout of FEX, there is an increase in FGF23 and an increase in SFRP4, both of which contribute to hypophosphatemia seen in XLH. So in tumor-induced osteomalacia, you have excessive production of FGF23 and SFRP4. This overwhelms the processes that normally are responsible for the degradation of these products. High levels of these products are then present in the blood, and they inhibit renal phosphate transport, and they inhibit 1-alpha-hydroxylase activity. In XLH, or X-linked hypophosphatemia, for reasons that we don't know about, there is increased production of FGF23 in osteoblasts. This FGF23 then enters the circulation, and it is possible that as a result of mutations in PEX, there is an alteration in the amount of FGF23 that is degraded, resulting in more FGF23 being around, and a reduction in 25-hydroxyvitamin D3 synthesis of 125-dihydroxyvitamin D3. Now, in autosomal-dominant hypophosphatemic rickets, Ekans and his group have shown very nicely that there is a mutation in FGF23 that prevents its further degradation and disappearance from serum that results in high amounts of mutated FGF23 in the serum and the generation of a phenotype. So there is a resistant peptide that is being generated and that is not processed in a normal way that is responsible for autosomal-dominant hypophosphatemic rickets. Now, in autosomal-recessive hypophosphatemic rickets, there are mutations in an enzyme or in a substance called DMP. There are also mutations in ENPP1 that can sometimes occur after the initial presentation of ENPP1 deficiency disappears, and there are sporadic cases of hypophosphatemic rickets due to the excessive production of clotho or of FGF23 for reasons that we don't understand. Now, the converse of hypophosphatemic rickets occurs in tumoral calcinosis, and in tumoral calcinosis, there are insufficient amounts of FGF23 being produced. The mutant FGF23 is not secreted and the individuals then develop hyperphosphatemia and high 125-dihydroxyvitamin D activity. Defects in the glycosylation of FGF23 cause the same syndrome, and also impaired activity of FGF23 can be due to a lack of clotho, which is the co-receptor for FGF23. Now, the next thing one needs to consider is whether or not the phosphotonins are physiological regulators of phosphate homeostasis. I think we can see, based on all the data that I presented on various disease states, that these are very important in the regulation of phosphate transport. But there are other factors that come into play in terms of regulating phosphate that appear to play a role in the first few minutes after one takes a high-phosphate meal. So, thinking about this, the chronic increase in phosphate or changes in phosphate absorption occur as a result of increases or decreases in PTH and phosphotonin, as shown on the right of the slide. But there are also changes in phosphate absorption that occur independent of PTH and the phosphotonins that occur very rapidly after one takes a high-phosphate meal. So, shown here is some work done from my laboratory in which we infused phosphate into the intestine of an animal. And shown on the uppermost slide is the fact that TMP by GFR, or the fractional excretion of phosphate, which is the opposite of the TMP by GFR, changes after you infuse phosphate into the duodenum. This occurs whether PTH is present or not. It's just that the baseline is changed a little bit when PTH is removed following parathyroidectomy. Now, these factors have allowed us to populate the presence of what they call intestinal phosphotonins. These are probably proteins. And these are responsible for the changes in serum phosphate that occur following a high-protein meal or the excretion of phosphate in the kidney following a high-phosphate meal in the short term. So one needs to think of phosphate regulation as both short-term and long-term. And we think that intestinal signals regulate phosphate in a feed-forward manner so that the increase in phosphate causes a change in phosphate handling in the kidney, as opposed to changes that are feedback control mechanisms that apply for FGF23. OK. So in the last few minutes, I'd like to talk about a clinical approach to hypophosphatemia and the use of antibodies against FGF23 in the treatment of patients with hypophosphatemia due to tumor-induced osteomalacia or due to XLH. My comments will be restricted mainly to tumor-induced osteomalacia, although much of what I have to say also applies towards the treatment of XLH. So when you see a patient with hypophosphatemia, these are the sorts of things you need to ask yourself. Is the disturbance acute or chronic? At what age was the change in phosphorus first noted? What's the family history? Are there bone or other physical abnormalities, at least in terms of growth, present in these individuals? And then one needs to make an assessment of what organ is responsible for the negative phosphate homeostasis by measuring the TMP by GFR and seeing whether or not the kidney might be the main player in the negative phosphate balance that is observed. And then one needs to think about whether appropriate counter-regulatory mechanisms have occurred. So one needs to measure a series of laboratory values that are shown here. They include minerals, alkaline phosphatase, creatinine, vitamin D metabolites, FGF23, SFRP4, and MEPI. And one needs to make an assessment of the efficiency of the kidney in being able to retain phosphate. Then one needs to do a number of skeletal investigations to try and localize the presence of a tumor that might be responsible for this particular syndrome. Let me just say that many of these tumors are very small and are not easily detected. And one needs to use a combination of scanning, as well as whole body magnetic resonance imaging, to localize the tumor. Some individuals have used venous sampling of FGF23 to try and localize an organ in which the tumor might be present. But we have not found this very useful, although others have reported it to be of value. Now, in this slide is shown a matrix of changes in various hormones that may occur following hypophosphatemia that occurs in different conditions. This is in your syllabus. And I won't go through every one of them. But I think you can discern by looking at this that changes in serum phosphorus are due to either associated changes in PTH or changes in 125 dihydroxyvitamin D. And one can distinguish various forms of hypophosphatemia from one another. So for example, in the case of nutritional phosphate deficiency, serum phosphorus concentrations are low. PTH levels are low. In the case of nutritional vitamin D deficiency, you have a low serum phosphate, but high PTH as a result of hypocalcemia that is causing hyperparathyroidism. And a combination of a deficiency in phosphate absorption that is vitamin D mediated in the intestine, as well as an increase in PTH, are responsible for the losses of phosphate that occur in the kidney, as noted by the change in the TMP by GFR. OK, so how do you treat a patient with TIO or inherited rickets? Until previously, until a few years ago, the only modalities of therapy that were available to you included the administration of phosphate and the administration of calcitriol. Calcitriol and phosphate can cause hyperphosphatemia transiently and can reduce renal function. And in not every case is there a resolution of many of the symptoms associated with hypophosphatemia, namely bone pain. And what I'd like to show you in the last few minutes is some of the work that we have done with a monoclonal humanized antibody against FGF23 in patients who have tumor-induced osteomalacia. And this shows that the administration of the antibody is associated with an increase in serum phosphate concentrations, a change in the TMP by GFR, and a change in many of the symptoms that are associated with this particular syndrome. Alkaline phosphatase and bone turnover markers change in a predictable kind of way. And fracture healing is greatly improved, both at 24 months and now in a long-term study at 144 months after administration of the antibody. So in conclusion, we can say that the administration of borosumab, sufficient to bring serum phosphate concentrations back into the normal range, is associated with the correction of many of the pathophysiological changes that are associated with tumor-induced osteomalacia and also with XLH. And I think that one should consider the usage of borosumab to treat hypophosphatemia in patients with tumor-induced osteomalacia who have tumors that are non-resectable, and also the treatment of XLH, both to restore growth, serum phosphate concentrations, and take care of many of the skeletal manifestations of the disease. So I think I'll stop here and take any questions. Thank you very much. Thank you, Dr. Kumar. Please join me. We have a few minutes for questions. Please walk up to the microphones. While people get ready, I'll start with the first question. You mentioned the parallel increase in FGF23 and SFRP4 in cases of TIO, for example. What is the clinical value of measuring SFRP4 in the case of TIO? What is the clinical value of measuring SFRP4? And do we have any clinical available assays? Right, right. Well, I think you get to a very key point. I think the majority of the pathophysiology of the disease can be explained by an increase in FGF23. It's only in a few cases where FGF23 is not increased that measuring SFRP4 is of value. Now, all the tests that I have on that grid are available at Mayo Medical Laboratories. You can't get SFRP4 measurements done routinely. You need to call somebody in my lab to measure them. But I think 99%, I would say 95% of patients with tumor-induced osteomalacia have disease due to FGF23 and not due to SFRP4. It's only in the few cases where you can't explain it that measuring SFRP4 and MEPI is of value. Thank you. We'll start with the first question. Dr. Wormuth? Yeah, thanks, Raj. That was great. I have a question about FGF23 levels that are normal or even high normal with hypophosphatemia where you see phosphate wasting. We've seen some of these patients where they're not actually elevated with their FGF23. Well, I'm not sure exactly what that might be due to, but it could be due to a change in processing of the hormone. So you need full-length FGF23 in order to get a given effect on phosphate trend. If, indeed, there are changes in the efficiency with which FGF23 is processed, you may get alterations in the efficiency of phosphate absorption. There could be other factors that are responsible, too. I just don't know what they are. Thank you. Next question? Giuseppe Berbezina from Boston. Thank you for that great overview. I was wondering, you didn't mention the use of burosumab in ADHR. Is that because the mutated FGF23 will not react with that antibody? Well, I suppose you could use it. I just don't think there have been enough studies done or enough patients examined for us to make any kinds of conclusions. I think the commonest causes of hypophosphatemia, of inherited hypophosphatemia, are XLH, where FGF23 is elevated. And I think nice long-term studies have been done. In the case of the autosomal-dominant, autosomal-recessive hypophosphatemia crickets, although FGF23 may be increased, there are a number of other mechanisms that could be responsible for the hypophosphatemia. I think it's something worth trying. If you have a patient, giving them burosumab would be something that you could do. Have you had an opportunity to look at that? Not really, but I was just curious about why it wasn't theoretically a target for the antibody, given that FGF23 is, in ADHR, the mechanism, right? Right. Well, the antibody should work, OK? I mean, the exact epitopes for the antibody are not known. But I think both in the case of ADHR and AHR, the recessive form of hypophosphatemia crickets, one would think that burosumab could work. And I think it would be worth examining. Thank you. Next question. Sudhir Bansal from Rhode Island. Number one, I'm very happy to see you, Rajiv. This has been 52 years or so since I was a year ahead of you in the same medical school. And you are really a great scholar, obviously. And we appreciate what you did. The presentation was very nice. I happened to have a case with male hypophosphatemic inherited syndrome. But there was no family history available. The mother had died. He only had a sister. He never had children. He did not have the genetic variation, but had all the manifestations with the FGF23 levels and the 125, and actually was totally disabled and did very well with the burosumab. And since this was so long lasting, we did not have, as a clinician, we just suspected that he did not have a tumor making. After making that comment, this person also had cardiovascular disease very early in his 40s before I had seen him in his 50s. In fact, I waited for burosumab to come out to start implementing therapy with him. Incidentally, Carl Insania was so everybody's senior, junior to me, did very well, incidentally. Carl Insania, who did the excellent hypophosphatemic studies from Yale, was a year junior to me in the fellowship program. So maybe everybody should be junior to me. They do much better. Anyway, so what I was raising a question was, was there any implication with the fibroblastic growth factor in terms of cardiovascular disease in this population? Because this has such a raised level of the fibroblastic. Right. Well, I think you raise an important point. I think people have shown that as renal failure progressives, FGF23 concentrations increase. And cardiovascular disease that increases in frequency and prevalence with chronic renal failure is associated with an increase in FGF23. So there is indirect evidence to suggest that there's an association between high FGF23 concentrations and cardiovascular disease. In our patients and in the XLH trial, when you gave burosumab, we did look at echocardiographic cardiac output parameters in these individuals. And there was no change. Other people have done studies in which they have made transgenic FGF23 mice and have looked at cardiac function before and after administration of FGF23 and have not found anything. So I think it's a good point that you raise as to whether or not there's an association between FGF23 and cardiovascular disease. So far, my own feeling is that it's an association. I don't think causality has been proven. Thank you. Thank you for your comments about my being around for such a long time. I was joking with my colleagues. I feel like a little bit of a dinosaur, but there you go. Fadi Al-Khair, Connecticut. Thank you very much for the wonderful talk, which is also great for folks preparing for their boards. Allow me to ask a question not related to what you discussed with us, if I can. Pseudo-hypoparathyroidism, can I ask a question about that? Sure, you can ask. I'm not sure I'll give you a good answer. Just a patient I got five weeks ago, the hospital called me, significant hypocalcemia, 19 years old. From history, she had these symptoms of titania as a kid. Never been diagnosed. Short, long story short, I stabilized her. And as outpatient, she's doing well. The only clinical diagnosis that I made is pseudo-hypoparathyroidism. Genas, test came negative. Genetic test came negative for the gene responsible for the pseudo-hypoparathyroidism. Is there any suggestion, like I can do the test at a different institution, or what comes to mind when you suspect this condition, but genetically you cannot make the diagnosis or confirm the diagnosis? Well, I'm not sure that I can help you, but I do know that there are a number of different mutations and different transporters that occur that are responsible for hypophosphatemia. And sometimes you can find those if you look. So for example, NAPI 3 mutations are sometimes associated with hypophosphatemia as well. This patient was hyperphosphatemia. She had actually significantly high intact PTH, significant low calcium, high phosphorous level. Right. Well, I think short of sequencing the FGF23 gene, I can't think of any other ways that you would look for a mutation that might be responsible for tumoral calcinosis. Now, in the classical cases of tumoral calcinosis, there is a difference between the ratio of full-length FGF23 and fragments of FGF23. Tumoral calcinosis, I thought, would be associated with normal calcium, normal intact PTH. My patient had significant hypocalcemia and significant high intact PTH. That's why it did not come to my mind as familiar. So when I did my own search, honestly, with this combination, I made the diagnosis of pseudohypopara. Not that I'm an expert, but I have only one patient currently I treat, which was confirmed to me. And I thought this would be my second case. With calcitriol treatment, calcium, vitamin D, she's doing very well. I mean, she's so happy. She's a student at UConn. And she told me this is the first time she does her exam without having these, like, you know, they diagnosed her as a kid being anxious, anxiety, having the contractions in her hands and all of it, which happened mostly during exam spells. I'm sorry, we're out of time. We can continue this discussion offline. Thank you. Thank you, everyone, for joining. And thanks again, Dr. Kumar, for your talk. Well, thank you.

phosphatonins

regulating phosphate homeostasis

tumor-induced osteomalacia

X-linked hypophosphatemia

autosomal-dominant hypophosphatemic rickets

FGF23

phosphate transport

hypophosphatemia

burosamab

bone mineralization