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[00:00:02] Dr. Joseph Muenzer : So, welcome. It's my pleasure to give a lecture on mucopolysaccharidosis, a primer for the pediatrician. I'm Dr. Joseph Muenzer from the University of North Carolina in Chapel Hill. I have the following disclosures to give. I consult for a variety of pharmaceutical companies. I serve on advisory boards for the same companies, and I also am principal investigator for clinical trials on intrathecal enzyme replacement therapy, and soon to be a gene- editing clinical trial for MPS II. The goal of this lecture is to give an overview of the MPS disorders, to talk about the clinical spectrum and the biochemistry of each of the MPS disorders, present some of the common clinical features, and at the end talk about the current treatment options. So in terms of the MPSs, they're a group of lysosomal store diseases. There's 11 known enzyme deficiencies comprising seven different clinical types. Each disorder is very rare, but as an aggregate, about 1 in 25, 000 patients in North America have an MPS disorder. The hallmark of these disorders is really the tissue storage of glycosaminoglycans due to the deficiency of the lysosomal enzyme. This results in progressive clinical disease with multi- system involvement, and there's a wide range of clinical involvement within each MPS, and there's also a wide range among the MPSs.
[00:01:25] Dr. Joseph Muenzer : This composite slide just shows an example of that. Every one of these individuals have an MPS disorder, from an adult with MPS II to another adult with MPS I, to a child with MPS I in terms of Hurler's syndrome, to a Sanfilippo, to another Sanfilippo, to an individual with Morquio, to another Morquio patient, and to another MPS II patient. One of the challenges for the MPSs is really the nomenclature. Historically, Hurler's syndrome used to equal MPS. And then there was the Hunter form of Hurler's syndrome, but now we reserve the term Hurler's syndrome really for the severe form of MPS1. Both MPS I and MPS II are single enzyme deficiencies, and they both result in storage of dermatan and heparan sulfate, where in contrast, MPS III has four different enzyme deficiencies, all resulting due to the storage of heparan sulfate, and it's a primary neurologic disease. MPS I, II, and III are by far the most common forms of MPS in North America, where in contrast, Morquio, MPS IV, there's two different enzyme deficiencies, it's a much rarer form. Morquio A is much more common than Morquio B. The hallmark of the Morquio is really a skeletal dysplasia due to keratan sulfate storage. MPS VI, or Maroteaux–Lamy, it stores dermatan sulfate, and that's a disorder without neurological involvement, but lots of physical involvement, and Sly's syndrome, or MPS VII, is one of the rare MPS disorders very similar to MPS I in terms of a range of clinical involvement due to b-glucuronidase stiffness of DNA, and that's all about dermatan and heparan sulfate storage. Recently, we've added a new disorder to the MPS list, MPS IX, due to hyaluronidase. There's a few patients reported with this disorder, primarily with joint involvement, and I won't speak any more about MPS IX. What is the rationale for this wide range of heterogeneity? Part of it's really due to what's being stored. So different compounds are stored in different of the MPSs, so dermatan sulfate is primarily located and made in the peripheral tissue or somatic tissues, heparan sulfate is primarily found in the brain or neurologic, and keratan sulfate is in the bone. So when you just have keratan sulfate, as in Morquio syndrome, MPS IVA, you have primarily a bone disease, where in contrast in MPS VI, where you only store dermatan sulfate, you have physical disease but not the neurologic disease you see in some of the others, and that's because in MPS VI you don't store heparan sulfate, which primarily occurs in the central nervous system. Sanfilippo syndrome only stores heparan sulfate as an example of a progressive neurologic disorder without any other involvement. What's the biochemistry of the MPSs? They're all due to enzyme deficiencies involved in the breakdown of glycosaminoglycans. The uniqueness is that these enzymes only work on the non- reducing terminal ends of the repeating disaccharides called glycosaminoglycans. Here's an example where you're missing the MPS I enzyme. You can't cleave an ironic acid off, and therefore the rest of the chain does not get broken down and you result in storage of that particular compound. When you have storage, you have what on the right- hand side where you have a lysosomal storage of these fibroblasts. If this was an MPS, it was restored with glycosaminoglycans , all of the clear areas are distended, lysosomes filled with storage materials that get washed out in the fixative process. This occurs, this cell no longer can function as well as it can. You have enough of this, the cell can die, and this results in the progressive problems we see in the MPS patients. Let's now talk about the clinical features we see. So MPS I deficiency of the enzyme iduronidase onset occurs prior to six months old age in a severe form of Hurler syndrome, and these patients with untreated will die between three and ten years of age of overwhelming both neurological and physical disease. It's a rare disorder estimated incidence of one in 100, 000 in autosomal recessive. So the young man on the right, Chris, is a four- year- old here and has a lot of the typical features of an MPS I Hurler child, the prominent forehead with macrocephaly, depressed nasal bridge, prominent tongue, basically no neck, stiff joints in terms that he can't extend his arms fully. Same way with the hips, hepatomegaly if he had a side view, and already he's significantly developmentally delayed in this picture. He died a few years later after a viral illness of a sudden death most likely due to coronary artery disease. Not just a severe form of Hurler syndrome, there's a whole spectrum of patients who are much milder. Initially Scheie syndrome used to be called MPS V, but my mentor Liz Neufeld recognized that that was really the mild form of MPS I or Hurler syndrome, and so now we have a whole spectrum of patients. It's important to note that all these patients have undetectable enzyme activity. So historically we talked about severe, intermediate, mild going along with the three forms Hurler, Hurler-Scheie, or Scheie. People thought these were unique disorders, but they're really a continuum between severe and mild, and so because of that people renamed it from severe to attenuated, showing more of the spectrum of disease, and there's patients that fall betweenHurler, Hurler-Scheie, or Scheie and it's sometimes very difficult to recognize who has what clinically. This is an example of the disease progression we know has occurred from the progressive storage of glycanate glycans.
[00:07:07] Dr. Joseph Muenzer : Here's a 10- month- old with Hurler syndrome with a severe form of MPS I, and that picture really does not convey that she has a life- threatening disorder. We're down here at 39 months, it's pretty obvious that she has a storage disorder. One of the other challenges that even within the intermediate or Hurler- Scheie phenotype there's a wide range of clinical involvement from patients who have severe joint disease to very mild joint disease, to patients who have significant airway disease, who have no sleep apnea, to massive liver enlargement, to really moderate liver enlargement. So this is a challenge within the disorder. There's a lot of heterogeneity even within one subtype of MPS I. In terms of genotype- phenotype correlation, there is, since there's a lot of known in the European ancestry patients, non- sense mutations that are very common that these – if two of these occur, either homozygosity or compound heterozygosity, they always have severe disease. There are other patients with missense, a lot of them have attenuated disease. So there's an example of another missense mutation, P533R, where you can have both severe and attenuated. So genotype- phenotype cannot always predict, and one of the biggest challenges we have in the future is there's a lot of pseudo- deficiencies, particularly in the U. S. among the African- American population that are going to be picked up by newborn screening but have no clinical disease. So switching now to MPS II, deficiency of idurinate-2-sulfatase, also called Hunter's syndrome. In this case, the mild form of the disease did not get identified until after the biochemistry came along, and so we don't have a different name for the attenuated form of Hunter's syndrome. Patients with severe form of Hunter's syndrome typically get diagnosed between one and three years of age in the severe form, a little later than the severe Hurler patient or severe MPS I or Hurler patient. Patients will die very premature typically, untreated in their teenage years of overwhelming neurologic disease, superimposed, and cardiac and airway disease. It's an X- linked disorder. Carrier females in general have no evidence of clinical disease, though there is a small number of females reported due to X autosomal translocations and other nonrandom X inactivations. So even though you have a patient who is a female, they can have Hunter's syndrome even though it's very rare. As we've seen in MPS I, there's clearly a spectrum from the severe to attenuated. Here's a patient, Craig, who is profoundly impaired in this picture, onset of disease in the first year, two, or three years of life. He became cognitively impaired. He died in his late teenage years, and here's a young man in his, what, 45 in this picture, 40 in this picture, intellectually intact, now at age 65 plus has some evidence of airway and cardiac involvement but still has normal cognitive function. We clearly have patients in between this two, but in general in Hunter's syndrome there's pretty more dichotomy between severe and attenuated compared to MPS . Molecular genetics of MPS II. Located on the X chromosome, XQ27. 3Q28, there's a lot of genomic deletion and rearrangements and these patients always have severe disease, but the big challenge for MPS II is most families have private mutations and we cannot predict disease severity based on genotype. Now switching from MPS II to Sanfilippo syndrome or MPS III, here's a typical picture of a Sanfilippo child who has very mild coarse facial features and some stiff hair, but there's four different enzymes all have the same clinical manifestations. They develop profound mental deterioration, they're very hyperactive, they have relatively mild to no physical features, and again like the MPS II patients die in their teenage years of overwhelming neurologic disease if untreated. We probably miss the milder form because we don't recognize them with just mild developmental delay, and so there are probably Sanfilippo patients out there who have it, but in their teenage years we don't know about it. Here's some other pictures of an individual with Sanfilippo syndrome. Here's a young man, quite known years ago, who actually died in his early teenage years because he actually liked to chew a lot of things as a mouse and he actually aspirated on a balloon in a care facility. So Morquio syndrome has normal intellect due to two different enzyme deficiencies. It's a much rarer disorder in North America and the primary problem in Morquio is really bone involvement and it's autosomal recessive. One of the hallmarks of the Morquio syndrome compared to the other MPSs, the MPS I and II, have very stiff joints with time, and you'll see that later, where the Morquio patients have joint laxity. They also have, we'll talk about later, an issue in terms of cervical cord instability, and so a number of them are in a wheelchair due to that and or because of severe hip disease, as we'll talk about. Severe short stature occurs with these patients. So MPS VI, in contrast to Morchio, is primarily a physical disorder due to deficiency of arylsulfatase B, multi- system involvement, but they have normal intellect. They die very premature in the first and second decades of life of overwhelming cardiac and airway disease, and it's an autosomal recessive disorder. Here's an example of just a spectrum we see of all the MPS disorders from patients who are much more physically involved to somebody if you didn't, if you look at his face you would say he's normal, but he had a family history, so he was recognized much sooner than he would have otherwise been. And so it's really this rapid or slowly progressive sort of somehow to define some of the severities of the disorder. One of the rare disorders is Sly syndrome, due to deficiency of β-glucuronidase deficiency. In this disorder, hydrops fetalis or nonimmune hydrops fetalis is a common presentation. These patients are very similar, patients who present later on in life to MPS I with both physical and CNS involvement, a very rare disorder in North America, much more common in other parts of the world as also with MPS VI, and it's an autosomal recessive disorder. So as we saw, there's a spectrum of disease in the MPSs from the severe to attenuated. I just alluded to hydrops fetalis as a common presentation of MPS VII. MPS I, MPS II, and MPS VII all have multisystemic involvement and CNS involvement, but never only have CNS disease. Where, in contrast, Sanfilippo syndromes only have primary CNS disease and the mild forms we most likely mix. We don't detect. MPS IV is really a skeletal disorder with normal intellect, and MPS VI, again, is a multisystemic somatic involvement but with normal intelligence. So what are some of the early clinical features suggestive of an MPS disorder?
[00:14:15] Dr. Joseph Muenzer : I should note that most MPS individuals appear normal at birth, except for the rare Sly patients, but otherwise. One of the challenges that, for example, things like inguinal hernia, hearing loss, recurrent ear infection, delayed speech, noisy breathing, all can occur in a variety of ways in a typical developing child, but when you see more than one of these features, that's when the issue of suggestive of an MPS disorder is really important. So once you see decreased joint range of motion, hepatomegaly, kyphosis, those alone should sort of say this is a possible MPS, but if you, for example, see inguinal hernia and hearing loss or inguinal hernia and delayed speech, that should prompt you to have multiple systems involved and think of a genetic disorder. The major clinical manifestations are alluded to virtually occur in every organ of the body. The two systems we don't have on the list is kidney, MPS patients do not have kidney involvements, and even though they have hepatomegaly due to storage, we do not see liver dysfunction. So what I want to do next is really talk about some of these features in terms of, I'm going to cover neurologic, cardiovascular, pulmonary, musculoskeletal, and vision, all as an example of the different features we can see, and so now talk in terms of neurologic disease in the MPS patient. This is caused by storage in the neurons, macrophages and meninges. What we see is this progressive cognitive impairment in severe patients and in these MPS II patients. We also see a communicating hydrocephalus that is unique for pediatric patients. Since most hydrocephalus in pediatric patients is obstructive, this case is the communicating. We see spinal cord compression due to thickening of the dura. We also have carpal tunnel syndrome involvement, which is a very unusual pediatric problem, but when you see it in pediatrics, it's almost always associated with the MPS patient. Here's an example of communicating hydrocephalus, as we all know, fluid is made along the walls of the ventricle, flows through the third and fourth ventricle, typically comes up over the top of the convexity and gets reabsorbed in the arachnoid granulation. It's believed to be that this reabsorption is not working properly in the MPS patients who have it, typically MPS I and MPS II, MPS VI and MPS VII. One of the challenges is how to determine that. Certainly if you see an MRI like this, you know this is obvious ventricular amygdalae in an MPS I patient, that patient had very elevated intracranial pressure. One can also do a spinal tap or a lumbar puncture, measure the opening pressure, typically done under anesthesia, has its challenges, another way to assess whether you have an increase in intracranial pressure. One of the other major concerns for the MPS patients with physical involvement and neurological involvement is spinal cord compression. Here's an example of an MPS I patient, a teenager, with very narrow or no fluid in the cervical region. The fluid is the gray of the spinal cord, you see complete loss of any fluid around that. This is not due to bony involvement, but due to dura. The dura can be as thick as cardboard, 3, 4, 5 millimeters thick and can cause a cervical myelopathy if untreated. Carpal tunnel syndrome in MPS I, II, and VII occurs and is typically underdiagnosed. One of the reasons it's underdiagnosed is that the symptoms typically in the MPS occurs only occur when you have significant reduction of nerve velocity, so only when you have profound involvement do they have symptoms in contrast to the adults with carpal tunnel. Very early on you'll have symptoms, so it's very challenging. One has to almost do nerve conduction studies prophylactically in terms of trying to figure out who has it what before they actually ever complain. Curly surgery is available in terms of standard surgery. The challenge of these patients have much more typical involvement due to storage around the carpal tunnel, and it's important to have some experience doing the procedure. Now I want to switch to cardiac disease, which is a common problem for the patients with the physical involvement. It's due to storage in the heart valves, coronary arteries, and aorta. Typically in MPS I patients, this is severe MPS I patients or Hurler syndrome. Historically a lot of them used to die after a mild illness. What happened, they tried to increase their cardiac output and actually had an MI due to coronary artery disease. Most of the other disorders where they have somatic involvement, it's really primary valvular disease, and on the right- hand side here shows a valve from an MPS I patient post- mortem and just showing how thick and abnormal that valve is. These valves should be just paper- thin leaflets and it's hard to even recognize which valve that is. All this can result in both cardiomyopathy and I find treated congestive heart failure. So what's the pulmonary disease? This is another severe area of issues caused by storage in typically lung, airway epithelium, and bone. Some of the major clinical features are obstructive airway disease, restrictive lung disease due to small ribcages and stiff joints that just can't take a normal breath. They have decreased diaphragmatic excursion due to hepatomegaly in the untreated patients. This results in frequent infections with thick secretions, which compromises them, all then resulting in severe respiratory insufficiency. Here's an MPS I patient, an x- ray showing the skeletal disease, the prominent oar-shaped ribs. This case this individual has a brachiotomy because of the severe upper airway obstruction. That upper airway obstruction is caused by storage at multiple different levels in the airway and abnormal tracheal ring formation. You can see this individual here with a very prominent tongue. He also would have very prominent lymphoid tissues. The airway epithelial have increased storage along with soft tissue. There can be tracheal storage, but also, more importantly, tracheomalacia causing a very floppy airway contributing to their upper airway obstructive disease. The other major, sort of, one of the major organ systems involved is the musculoskeletal. It's caused by storage both in synovium, periarticular tissue, and bone. So in terms of joint restriction and stiffness, it's really a hallmark of the MPS I, MPS II, MPS VI, and MPS VII patients. They can have involvement of the hands, spines, and hips, and knees, and I'll show some of that. So here's just an example of both restriction in terms of the hips where this individual can't fully extend his knees and has significant limitation in hips, and here's a 13-year-old with Hunter syndrome who that's the best he can do in terms of raising his arms. He has enough shoulder restriction before onset of therapy. These individuals will develop untreated, a very claw-like hand which has really severe limitations, so a combination of the joint involvement and carpal tunnel syndrome can really have dramatic impairment of their quality of life. This particular individual could not eat a hamburger by one hand, actually had to take the hamburger, put it between his two hands and eat it that way because he couldn't pick it up to reach around it because of his limitations, for example. Spine disease is not uncommon. Here's an example of a Morquio with kyphosis showing significant curvature. Here's another example of a hip dysplasia, very abnormal femoral heads and acetabulum causing significant impairment, and that's why a lot of the Morquio patients can end up in a wheelchair just because they have such terrible hip disease. Hip dysplasias also occur in a variety of the other forms, and it's interesting that Sanflipo patients can be diagnosed with a hip dysplasia and think it's like Perthes disease, but in reality it's a Sanflippo hip dysplasia. Knock knees or genu valgum is very common in Morquio patients and some of the other MPS patients I'm showing here, and this is amenable to epiphyseal stapling. One of the other important features I alluded to for the musculoskeletal disease is odontoid hypoplasia and cervical instability are common. Acute spinal cord injury and or sudden death have been reported after relatively minor fall in some MPS patients, so it's important to get x- rays of extension flexion and make sure they have cervical stability. Particularly Morquio A patients have a high risk of developing cervical instability because of the ligamental laxity and odontoid hypoplasia. The last sort of major organ system we're going to talk about is the eye. Patients with MPS can get corneal clouding. MPS I patients occasionally have glaucoma. The vast majority of patients will get retinal disease in terms of a rod dystrophy, which means they can't see as well at night and have decreased peripheral vision resulting in really loss of blindness, but acute loss of blindness is not typically due to eye disease but increased intracranial pressure. So what's the disease pathophysiology in the MPSs? The deficiency of the lysosomal enzyme results in the GAG storage, but the storage is tissue-specific for each different type. So the liver and spleen enlargement we alluded to and the core spatial features you saw are really due to the tissue accumulation of the GAGs. The skeletal disease in contrast appears to be due to disruption of the growth plate where the heart valve disease appears primarily due to storage of GAGs in the heart valve and then secondary GAG, secondary fibrosis, and it's a good example that once you have that fibrosis all the enzymes in the world will not correct that. The airway disease is more complicated with secondary storage of GAGs in the soft tissues and the abnormal tracheal ring formation. But the real challenge we have in terms of the MPSs is the CNS disease. The etiology is really unclear with multiple possible secondary events. The GAG is a primary storage event, but what occurs next is unclear. There's inflammation, apoptosis, abnormal calcium metabolism, a variety of other mechanisms that are still being evolved and studied. So how do you diagnose MPS? It's clinical suspicion, clinical suspicion, clinical suspicion. If you don't think about it, you'll never make the diagnosis. So once you think about it, you can do x- rays looking for the bony changes that they see. One of the historically hallmarks was the increased urinary GAG analysis, and so a quantitative urine GAGs, and now we're doing component analysis of that as we can measure individually heparan, dermatan, and keratan sulfate by LC- MS- MS. Obviously the dye binding assays were very insensitive, and a number, for example, of the Sanflippo patients, 25% or so, would be missed because they didn't have enough material in their urine to be recognized.
[00:25:23] Dr. Joseph Muenzer : If you think about MPS or have suspicion based on x-ray or urine gags, then really the gold standard is do enzyme testing either on serum, white blood cells, or fibroblasts. So what's the treatment in terms of MPS? So enzymatic correction is possible at the cellular level in the MPS fibroblasts secondary to the following observations. So my mentor, Liz Neufeldt, recognized that cultured cells release small amounts of lysosomal enzymes. She initially called these correction factors, and then recognized later they're really due to the lysosomal enzymes coming out of cells. It's also important to realize there's a very efficient mannose- 6- phosphate receptor- mediated enzyme uptake that occurs in fibroblasts, and probably the other hallmark of this disorder is that you only need 1 or 2% residual enzyme activity to correct the GAG metabolism in a given cell. So you don't need a lot of enzyme, and that's why these disorders are much more amenable to treatment than others. So what are the current treatment options for these disorders? So one is hematopoietic stem cell transplantation, and also intravenous enzyme replacement therapy. So here's just a schematic of what happens to how a lysosomal enzyme is made. It's made in the rough endoplasmic reticulum. In the Golgi, a mannose- 6- phosphate gets placed on that enzyme. That allows it to be targeted through the clathrin- coated vesicles to the lysosome. But that process is not 100% perfect, and so some of the enzyme actually can leak out of the cell, and that enzyme can be recaptured because there's a mannose - 6- phosphate in the clathrin- coated pits and get taken to the lysosome or go to a bystanding cell. This is what happens with transplantation. In transplantation, your white cells now make a normal amount of enzyme, they circulate, they can release a small amount of cells and actually get correction of other cells nearby by a bystander effect, and it takes very little. So in terms of treatment, for MPS I, Hurler Syndrome, the treatment of choice is hematopoietic stem cell transplantation. In that disorder, the microglia cells of the brain are of bone marrow origin, so when you do a bone marrow transplant, you actually put new microglia cells in the brain, and with time you stabilize their progression. So we don't see the neurological progression that would occur in Hurler Syndrome with successful transplants.
[00:27:53] Dr. Joseph Muenzer : In contrast for the other MPS disorders, we can see physical benefit, but there's been really questionable neurological benefit for MPS II and MPS IIIA and B in terms of transplants. In contrast, we now have IV enzyme therapy available for both MPS I, II, IVA, and VI, and clinical trials are ongoing for VII and for IIIB in terms of enzyme replacement studies. So what happens with endo- replacement? Here's actually the first patient ever treated with endo- replacement therapy, it was an MPS I patient. He's roughly 17 to 18 years of age here, showing he's intellectually intact, but he has a tracheostomy. He has a stiff joint we talked about, hepatomegaly, can't really stand any more than that, can't stretch out more because of hip restriction and joint restriction. Here he is six months later. He has synthetic improvement, but he never got any better than that because of just residual disease. So even though he was better, he really had limited long- term benefit because he was started very late. In theory, if you can start somebody before they have lots of symptoms, enzyme replacement therapy is much more beneficial. So in terms of summary of IV enzyme replacement therapy, ERT and MPS results in reduction of urine gastric and decreased liver and spleen size across the board in all the MPSs being treated. We see improved joint range of motion, pulmonary function, energy and endurance occur in some patients after ERT. It's a fascinating phenomenon that a number of these patients actually feel better once they started on ERT where prior to that we had no appreciation, they didn't have the same degree of energy. There's no question in my mind that recombinant enzyme therapy appears to be beneficial for the somatic disease, it probably don't impact the skeleton eye in the MPSs, and that the IV ERT is not expected to help the central nervous system disease because of the blood- brain barrier. It just doesn't cross over. So currently we're really not treating the central nervous system disease in any of the other patients except for the MPS . There's a variety of clinical trials ongoing. I'm extensively involved in intrathecal enzyme replacement therapy where we're putting the missing enzyme directly into the spinal fluid. At the end of this year we'll have the results of a phase 2- 3 trial to see if that's working for Hunter syndrome. There's been intrathecal trials for other of the MPS disorders. There's currently an ongoing gene therapy trial for Sanfilippo syndrome both in Europe and the U. S. We'll be starting a gene editing clinical trial for MPS II and MPS I here and other sites in the U. S. There are ongoing trials in the U. S. of modifying the lysosomal enzymes so they cross the blood- brain barrier and those trials have some encouraging early results. Certainly anti- inflammatory therapy, stop codon read- through, and substrate reduction therapy are not yet in current trials but all will be in the near future. Thank you for your attention. Question? Yes, Dr. Muenzer, what are the challenges of early diagnosis in MPS? The question was what are some of the early challenges in diagnosing MPS? As I alluded to that prevention is really the name of the game in MPS. Children with MPS are born normal appearing and then slowly developing. That's one of the challenges. It's difficult over months to have that. Sometimes the families don't even appreciate that. I think the challenges are it's a rare disorder for people need to think when you have more than one system involved. For example, if you have hearing loss and you have hernias, one should think about that, but if you have even like a patomegaly and hearing involved, once you have more than one system involved, it's important to think about an MPS disorder. The rarity is really the challenge. In the U. S., a typical pediatrician may see one or two patients in a lifetime of practice. So one of the challenges in the future will be is to really develop newborn screening, to pick them up in the newborn period, do enzyme testing, determine who has what, and then start them on therapy appropriately and prevent some of their disease progression. You can get much better long- term outcomes. Thank you. Okay. If a pediatrician suspects that a child has an MPS disorder, what would their initial steps be? So the question was if a pediatrician suspects a child has an MPS disorder, what should they do? For me, if I saw that patient, it would be testing for urine GAGs, doing x- rays, evaluating the patient, and if I had any suspicion, then do enzyme testing. So a lot of people, that pediatrician normally would refer that patient in the U. S. to a genetic center that deal with those patients, and that would really facilitate that. Wouldn't expect the pediatrician themselves to work up that patient, even though they could, but it probably doesn't make sense. Because on the clinical presentation, I can get a sense for what type of MPS that patient may have. If I saw a patient who's a female with corneal clouding and stiff joints, who's a 2- year- old, I'd almost assume that's going to be a urler patient. I could focus my testing on that alone. Any other questions? How early can a patient begin enzyme therapy? So the question is how early can a patient begin enzyme therapy? Because of family history, we see actually young children who have an older sibling, and then when the next sibling comes along, we can diagnose them. You can actually start enzyme replacement therapy within a few weeks of life, if you so desire. Typically, four to six weeks, four to eight weeks, is probably not too early for some families. Can you have a diagnosis in vitro? So the question is, can you be diagnosed in vitro? You can be diagnosed, but typically only for the families who have a known child already affected, because again, prenatal diagnosis is available for all the MPSs, but you have to know what you're looking for, and typically these days we base it on DNA testing, where enzyme testing is much more challenging to do a prenatal diagnosis. So now with the availability of DNA testing, everybody should have, once they have a confirmed enzyme confirmation, have DNA testing to confirm what mutation the child has, and then that can be used for prenatal diagnosis. All right. Thank you. We're done? Thank you.