# Mucopolysaccharidosis (MPS): Understanding the Diagnostic Journey of Patience

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[00:00:00] Dr. Julie Eisengart: Well, hello, good morning, good afternoon, and good evening. Thank you very much for coming. My name is Julie Eisengart. It is my privilege and honor to be sharing the stage with Professor Roberto Giulgiani. We are going to be presenting on the early diagnosis of mucopolysaccharidosis, understanding the diagnostic journey of patients. Professor Giugliani is with the Department of Genetics at the Federal University of Rio Grande de Sul in the Medical Genetic Service at Hospital de Clínicas de Porto Alegre in Brazil. He is an incredibly well- known and an incredibly accomplished figure and mentor and leader in the field. He's a medical doctor with a specialization in medical genetics. He has obtained his MD and PhD degrees in Brazil and has had training all over the world. He's incredibly generous with his expertise and knowledge. He has been a founder and leader in multiple organizations and done incredible things for the field of genetics and certainly for the mucopolysaccharidosis disorders. He founded the Medical Genetic Service at his hospital. He's acted as president of the Brazilian Society of Clinical Genetics, the Latin American Network of Human Genetics, and the Latin American Society of Inborn Errors of Metabolism and Neonatal Screening. He's the director of the WHO Collaborating Center for the Development of Medical Genetics, and he's one of the top researchers and carers in the world in MPS disorders. So it is certainly a great honor and privilege to be sharing the stage. Before we begin and listen to what I'm sure is going to be an illuminating talk by Professor Giugliani, I have a few notes to prepare for this session. So for those who are kind enough to enjoy this session, please, we ask that you do not take any screenshots, nor please do not reproduce any of the slides, the content, or the images without the express written permission of the speaker. So today's format is gonna be about 50 minutes of presentation followed by a question and answer session via text. You can submit your questions at any point via the questions panel. They can actually only be submitted via text and unfortunately not verbally. We will take as many questions as possible, and those will be answered at the end. This webinar is going to be recorded and is going to be available online at the MPS Resource Center with the link available for your viewing. So thank you so much, and we will now begin with Professor Giugliani.

[00:03:14] Professor Roberto Giugliani: Thank you very much, Julie, for this extremely generous introduction. It is a pleasure and an honor for me to have you as moderator in this session. So I will talk about diagnosis on MPS. These are my disclosures. Okay. And so let's start with when to suspect MPS. One thing that we should always remember is that it's a genetic disease, so it's very wise to have a look in the family history. For instance, consanguinity in the family is something that may raise the possibility of a genetic disease. Take this into account. And we will remember that most are autosomal recessive, but there is one disease that is of this MPS that is X- linked recessive, so this is a disease that can occur in multiple generations. It's good to have a look on the pedigree. And also, always remind to ask for, when you are taking the family history, about the relatives that had some undiagnosed disease, and this also may be something related to MPS. So we will show in the next slide an example. So this is a family with the X- linked recessive MPS, that is MPS2 or Hunter syndrome. You see, this is the index case, the boy in the right with the mother. He is the index case that's diagnosed as MPS2. And at the beginning, he was diagnosed as an isolated case in the family. But when looking at the family history, we found that one uncle of this boy had a non- diagnosed disease. Then we asked some information from the family. They brought us the picture of the child, and having a look, you see that the same disease is also MPS2. So this is a disease that is X- linked recessive. So the mother is a carrier of this mutation. If the mother is a carrier, the daughter that is on the left may also be a carrier. So we need to have a look on this. And it has implications for genetic counseling. So this is the first thing, to remind that this is a genetic disease, MPS, and consanguinity in multiple cases in the family may occur. So next slide, then moving here. So the second thing is that usually, most cases of MPS, they are born as normal babies. The exception is that some cases may present hydrops fetalis. So even in pregnancy, we can have some information that- like we see in the left bottom side, we see a fetus with hydrops fetalis, and this may lead to the suspicion of not only MPS, but many lysosomal diseases may occur with hydrops fetalis. But if a child presents in the prenatal period, or you notice immediately after birth, think on MPS. Otherwise, most cases of MPS are normal babies in terms of presentation. The disease has usually a progressive course, a slowly progressive course. It is not like other genetic diseases that present as a catastrophic disease in the first months of life. No, it is usually a baby that is presenting some manifestation that takes some time to be noticed. And I will show you this example of this boy. This boy has, again, MPS2, the Hunter syndrome. You see, at two years of age in the left, he looks like a normal boy. Six years, you see that the faces are a little bit different. It's not a normal face, but it's not that striking manifestation. And you see in the right, the boy with 11 years, so the coarse faces, you see that it's a boy with a disease, and in fact, he died when he was 14 years old. So it's a slowly progressive course, but usually leading to reduced life expectancy. Another thing that is something, is the point, the situation that raises the diagnostic possibility is the visceromegaly. Usually, hepatomegaly or splenomegaly, or both, may be the first sign. But take care that some MPS may not present this, or maybe mildly form the MPS type 3, and maybe not occur at all in MPS4. But in most cases of MPS, they have this visceromegaly. So it's something that should raise the possibility of a lysosomal disease, including MPS. And this is an example that a girl with MPS type 1 having, you see, massive visceromegaly, prominent abdomen, also umbilical hernia due to this pressure in the abdomen. So this is quite common in the MPS, it may be the first sign. Coarse face is also something that may be the first characteristic to be noticed. It's not always present, again. Some cases of MPS4 and MPS3 may not present coarse faces, but many patients- this is a very young boy, around one year of age, you see the face is already with some coarse features, and sometimes it's the thing that will lead to this diagnostic suspicion. Joint stiffness is something that also, again, may be the first sign to be noticed, the especially in the hands, you see, but again, not always present in MPS3, may be not present in MPS4. In fact, instead of joint stiffness, they have joint laxity. But most cases of MPS have this joint stiffness, you see this hand, and this is something that also should raise the possibility of MPS. Bone dysplasia is something that sometimes they also occasionally notice when the child, for instance, does a chest x-ray due to a respiratory infection, and they notice some abnormalities in the vertebrae, in the ribs, and this may lead to the suspicion of MPS. But maybe quite typical, you call dysostosis multiplex, an abnormality that is common to many MPS types, like MPS1, MPS2, MPS6, MPS7. But in MPS4, you also have skeletal dysplasia, but the pattern is a little bit different. And in MPS3, maybe mild, these bone abnormalities, but something that you should have a look. You see the spine with the vertebrae that have these characteristics of the bone dysplasia of MPS. You see in the hands also the metacarpals and the fingers, they are not normal. They have some abnormalities, and the radiologists, they notice these, and sometimes it's the first thing that leads to the diagnosis. Corneal clouding, also quite characteristic, sometimes very early, sometimes takes a while to appear, in some cases maybe the first time. In fact, one type of MPS, the attenuated form of MPS1, it was described by an ophthalmologist because the patient was not very severely affected, but the adult presented corneal clouding, so sometimes it's the first time to be noticed. Depending again on the MPS type, it's maybe mild in the MPS3, and it's rare in the MPS2, despite this disease is quite severe, but it's not a feature usually of MPS2. Here is a corneal clouding that you see very intense. Respiratory infections, respiratory distress is common to most mucopolysaccharidosis, but these are normal, they are normal. This appears in many diseases, it's not something specific of MPS, but when you have all the data, usually you have also this respiratory infection, sometimes respiratory distress. Heart valve also is common on most MPSs, sometimes having consequences later in the childhood or early adulthood. And the previous surgery, this is something that is quite common in the MPS. Patients they have surgeries for hernia, for tonsils, place air tubes, adenoids, remove adenoids, but it's not suspect of MPS. But this is something that when you collect the history, this is very important. I will show an example, a study that we did in Brazil in over 100 patients with MPS, and you notice that 67 out of 113 had a surgery before diagnosis, so it's quite common to have surgeries. This is around 60% of all patients having at least one surgery before diagnosis, and some had more than one surgery. So it's something that also is important to notice. So one thing that is also common in some types of MPS, the severe form of MPS1, the severe form of MPS2, all cases of MPS3, and also MPS7, is the cognitive decline. But in some cases, and in some cases maybe the prominent feature of the disease. But this I will leave to Julie to describe better on her presentation. I will just move now to the diagnostic methods we have for MPS, so if you suspect MPS, you should request assay of urinary GAGs. So the GAGs are the glycosaminoglycans is the product that is stored in the mucopolysaccharidosis MPS, and these are quite well identified in urine. So you collect maybe a random sample of urine and you see the orange dots are case of MPS and the green line is the normal level. So we see most, if not all, patients with MPS have these increased GAGs in urine. But to be sure, you should avoid to collect diluted specimens. Notice that these numbers decrease with age, even in the normal people, so we should have age-related reference values, avoid diluted urine, and avoid these screening methods that you see in the right, methods that are just qualitative and maybe give false results. So we avoid these methods and ask for a quantitative assay of urinary GAGs. And if you have increased urinary GAGs and all the clinical features that I described before, this is suggest of MPS. Then you can classify your patient in one of these three main phenotypes. The phenotype you see in the left is a visceral disease with skeletal manifestations and usually they may have CNS components, and this is typical for MPS1, MPS2, MPS6, and MPS7. In the center you see a patient that is not so dramatically manifested, not so the physical manifestations, but has a neurodegenerative disease and this is typical for MPS3. And there are four types of MPS3, the A, B, C, and D, but this is the way you should investigate. And in the right you see a skeletal dysplasia that is different from the skeletal dysplasia you see in the left. And patients are normal in terms of neurocognitive development and they usually have the MPS4. So having this starting point, you have GAGs increased, you have the phenotype, then you have a look on the species of GAGs that you find, you identify in the urine. And then if you find, as you see in the left arrow, is the dermatosulfate and heparan sulfate, this is typical for MPS1, MPS2, or MPS7. If you find the keratosulfate, you see just that side of this is typical for MPS4. If you find only dermatosulfate, it's typical for MPS6 and so on. So it's only heparan sulfate is typical for MPS3. So now you have the increased GAGs, you have the clinical features, and you have the type of GAG that is stored. So you can go and now identify the enzyme that is deficient. There are 12 potential enzyme deficients in the MPS, but you will not need to assay 12 enzymes. You cannot say the enzymes that are most probably deficient, considering the phenotypes and the urinary GAGs. For instance, if you have a visceral megaliths, skeletal dysplasia, neurodegeneration, and you have presence of heparan and dermatosulfate in urine, GAGs are increased, then you can start assaying the enzyme for MPS1, the alpha-hydrolonidase. You do this by using in the blood, maybe blood spots, or maybe liquid blood, or maybe even plasma. You cannot say the enzyme using a method that you have an artificial substrate, that when the enzyme is active, it releases a fluorescent component and you measure the fluorescence in a standard fluorimeter, and you see the increase of the presence of this fluorescent substrate indicating normal activity. Or if you do not see the fluorescent substrate, you see that this is a deficient enzyme.

[00:18:52] Professor Roberto Giugliani: So this is a quite very standard method of measuring the enzyme activity. And so the usual processes, you start from clinical suspicion in the left, you go to the GAG analysis, quantitation, and also identification of the GAG species, you do the enzyme assay, and then you confirm doing the molecular genetics. Now you know which enzyme is deficient, then you go to the gene that quantifies that enzyme and identify the mutations. This is a standard flow of diagnosis. However, this is changing a little bit in the last- in the most recent time, because after we're having a clinical suspicion, you can go to- so many times you go directly to the molecular evaluation. You do a panel with all the MPS genes, and then you can find some variations that suggest some of the MPS. You can do a panel, or you can do a whole exome, or you can do a sequence or one gender to suspect that may be abnormal. Or if there is a family case, you can do the mutation present in that family. So there are different ways to make the molecular analysis, but also have the mutations, identify the mutations. You should confirm that the mutations have a functional consequence, so you go to the enzyme assay to check if the enzyme is really deficient. And even that, you should have- you should identify the GAGs that are increased, so you should confirm that that genetic variation, they are leading to enzyme deficiency, and that enzyme deficiency is leading to GAGs accumulation. Then you have the complete diagnosis. So this is what we need for complete the diagnosis. Again, this is an example, this is a girl. I suspect of MPS, I did the GAG quantitation, it was increased. I went to look at the type of GAGs, it was heparan sulfate, suspicion of MPS3. Then I took blood and did the assay of the enzymes that are deficient in MPS3 are four types of MPS3. So I assayed four enzymes and this was a case of MPS3C. Then I did the- in fact the MPS3B, the boxes are a little bit ... MPS3B. Then I did the specific, the sequencing of the gene related to MPS3B and found the mutation, so the diagnosis is complete. Okay. So I think this is- I just gave an overview about the clinical suspicion and how to make the diagnosis. We can maybe take some minutes to have some questions and have a discussion. Julie?

[00:22:00] Dr. Julie Eisengart: Thank you so much, that was a really wonderful and detailed walkthrough of the many ways to have suspicions and how to proceed with diagnosis, and informative indeed. I was struck by your work on tallying the surgeries before diagnosis. This is something quite striking. And I think about a lot of the conversations that have been had by MPS leaders, yourself included, about the concerns with airways and how delicate intubation can be for some MPSs, particularly for surgeries with the airway complications. And I'm curious if there is some knowledge about how the airways may progress and if in fact the anesthesiologist might be one of the people who become suspicious of MPS because of how the airways present in those early surgeries.

[00:23:06] Professor Roberto Giugliani: Well, yeah, it's something that usually came along other- it's rare that we have the respiratory manifestations and do not have the visceromegaly or coarse faces or joint inflections. So it's something that sometimes is what leads, takes you to the diagnosis, but it's something that you should also raise the need of checking other systems and usually you find other manifestations. But it is some kind- the family comes and say, "Oh, the child is snoring a lot," and this is something that's very common to have this snoring. Also his the sleep pattern is very disturbed because of these obstructions, obstructive disease. And so it's something very frequent, the impact on respiratory, and this is something that we will talk about treatment in another session, but it's something that is really improved with the therapies available for some of the MPSs.

[00:24:24] Dr. Julie Eisengart: Thank you. I'm also wondering, now that you mentioned the respiratory piece, how often there's hearing loss due to fluid and obstruction, is that universal to the MPSs or still selective to some types?

[00:24:46] Professor Roberto Giugliani: Julie, this is a very good point. I did not mention this because there are many things to mention on MPS, but this is one point that is really hearing loss is something that is very common in MPS type 2, but is present in most of MPS. It may be some mixed causes like a neurologic cause or maybe a conductive cause and this is something that- and this can lead also to speech delay and then also can influence the development of language, so it's something that we should- It's not something that takes to the diagnosis because usually the other are leading causes of taking you to diagnose, but it's something that is present in most cases, and if you investigate properly, you'll find some degree of hearing loss is really very common. Very good point, Julie, for bringing this.

[00:25:55] Dr. Julie Eisengart: Well, thank you very much. This is really rich information to be hearing straight from you. I think we might have time for one more question, if I may indulge, before we move on to the next piece. I'm curious as we talk about these, this focus on early diagnosis, and you've raised these helpful points about the parents raised concerns about the snoring and the disrupted sleep and maybe some hearing loss or some delays. When people are strategizing, who are the best clinical providers to target with this kind of information. What, in your impression, are some of the specialties who are most likely to raise concerns that perhaps MPS is at play when a child is being examined, for example, bone specialist or radiologist? I'm curious about what are some specialties in your opinion.

[00:27:12] Professor Roberto Giugliani: Yeah, very good point as well, because it's a multisystemic disease and, depending on each patient, there are some higher degree of involvement of maybe the heart or the viscera and then this. So the common point is that the pediatrician is the one that usually notice that something is going wrong. But then they usually ask the opinion of another specialist, and in the case of patients that have some neurocognitive involvement, the child neurologist is someone that is consulted very frequent. Sometimes, as you mentioned, the radiologist is the one that notice that there's something wrong sometimes as an incidental finding. Orthopedic surgeons are also very frequent to be consulted because of the bone problems and the joint restriction, and also the hepatologist or the gastroenterologist too because of the viscera megale if they increase more in the spleen than in the liver. Sometimes the hematologist is also consulted because of the increase in the spleen, sometimes thinking some malignancy or some- Or the pediatric surgeon also because of the hernia, and also the ear, nose and throat specialist also because of the tonsils, the adenoids and also the hearing and the ear tubes. And so there are many specialists that may be involved. Now, at the end, the patient goes to the geneticist, in our case, or the metabolic pediatrician, depending on the region you are or the availability of the specialist. But there are many specialists that are involved and of course the respiratory doctor at some point will be also involved on these cases. Also the neuropsychologist, because in many cases, as you probably will mention in your presentation, will have this need of doing some testing of the development. But it's important also to raise awareness about this disease on other health professionals, like nurses and people working in the health, primary health care, because they may notice that that family has a different condition that may be some genetic disease.

[00:30:13] Dr. Julie Eisengart: Well, thank you. It really is- and there's a whole host of opportunities to identify potential clinical signs, and I'm really glad to hear your expert talk on this so that this awareness can continue to be enriched and raised. So, thank you.

[00:30:34] Dr. Julie Eisengart: So I will be moving on to discuss how an early diagnosis can help improve the outcomes in MPS. I'm an associate professor of pediatrics with the University of Minnesota. My training is in pediatric neuropsychology. Here are my financial disclosures. Next slide, please. So today for my talk, I'm going to be highlighting how MPS affects function, what I mean by function, how we measure function in MPS, and then I'd like to spend some time talking about how outcomes can truly be affected by early diagnosis. Next slide, please. So when I say 'function', I am referring to the many ways that a person functions. So there's cognitive function. Many use this term to mean intelligence or IQ, but there are other aspects of function, too, that are very important to MPS, such as communication, attention, the ability to be independent, behavioral profiles, and so on. Next slide. In MPS, as Professor Giuliani was covering, there are so many aspects from head to toe of systems that are affected. And so he mentioned, of course, many of these pieces. But if I may, just to illustrate, talk about how sleep is disrupted because of upper respiratory issues. Maybe there's even central sleep apnea. And when sleep is disrupted, any one of us can relate to how a poor night's sleep might affect our function. So children who are having disrupted sleep may have irritability. They may have trouble focusing and learning. And so while it is caused by their MPS in a somatic sense, it may not necessarily be a precise, direct effect of MPS on the brain. And so this is true for many of these, such as the physical limitations that may, in turn, affect a child's ability to learn and interact with the environment, and so on. Next slide, please. So how do we measure function? I would like to spend a little bit of time talking about what we do in pediatric neuropsychology to really track how an individual with a type of MPS that may involve cognitive decline, how do we measure this? And so, as previously mentioned, the types that most often have cognitive decline are the neuronopathic forms of MPS1 and MPS2, as well as all forms of MPS3 and MPS7. So I have here a hypothetical illustration of how a disease may affect development. And on the bottom axis, we see the chronological age, and on the y-axis, we see the mental age, which is sort of how the person is functioning, what's their age level mentally? Next slide. So here, I've highlighted what is the vector of normal development, and that is where the chronological age and the mental age are the same. So a two-year-old is functioning at a two-year-old level, a four-year-old is functioning at a four-year-old level, and so on. However, in this hypothetical illustration, we also have the vector of disease effect. So this is the force of disease on the child's functioning, and you see it's in a downward angle. Next slide, please. And so I've highlighted here that downward vector, and you see that it crosses at about age four. So what this means is that even though this disease has been evident from birth, the pathological cascades of the accumulating disease substrate don't add up enough to overtake development until, in this case, about four years old. Next slide, please. And so I've highlighted here what happens to the child's development when the vector of disease has finally crossed at age four, begins to pull the child away from normal. So next slide, please. Here, I've highlighted this very challenging point where the disease has begun to pull the child away from the normal pace of development, yet the child is still developing. And so there are skills being gained, just not as fast as unaffected peers. And if you intervene during this time when there is still the ability to acquire skills- next slide, please- such as right here, if there is sort of an effective therapy- next slide, please- what we'll see is that the child is able to continue on if the therapy is effective for the child, is able to continue on with development. Now, on purpose, I've made that yellow line not parallel to normal development. So there's forward progress, but it might still be slower than an unaffected child. However, if we look further on that resultant vector of development in an effective child- next slide, please. And we focus on this area where the child has actually begun to lose skills and have some regression or the actual cognitive decline. If we intervene there, then- next slide, please- it's not as obvious- it's not as certain what the outcome will be. Will there be just sort of a freezing in time, a stabilization of skills? Will, unfortunately, skills continue to regress or will there be some ability to learn? Next slide, please. So now that we've sort of gone over the way that we have been measuring and tracking, I'd like to present to you the different paces of development and decline in a few of the MPS types. So on the left, we have the neuronopathic form of MPS1, that's Hurler syndrome. And we see, again, this is that same format of some early development and then some slowing down in the skill acquisition and eventually decline. In the center, we have an assembly of three different natural history studies of the neuronopathic form of MPS2, Hunter syndrome, and we see they follow kind of a similar course. The beginning of the slowing down of skill acquisition occurs a little bit later than can be measured in Hurler syndrome. And then in MPS3A, as Professor Giuliani mentioned, there are three forms of- sorry, four forms of MPS3. MPS3A is the most plentiful, and here is an integration of multiple natural history studies that shows kind of a later period of decline, but always developmental delays. Next slide, please. So I probably don't need to go too deeply into this because this has been so expertly covered, but really, there's a very significant challenge of detecting MPS conditions early because many of them have nonspecific symptoms. Next slide, please. Really, the ideal time in terms of neurocognitive and functional outcomes is to be intervening before there's any change to the developmental trajectory from kind of an unaffected child. So that's in this green highlighted area. The problem is that in that green highlighted area, it's a very difficult condition to discern from other typical, more common, and less lethal childhood conditions. Next slide, please. And here, what I have are three children with different MPS types in the early stages, and these children, you could argue, are in that area of the disease trajectory that I just highlighted in green when it is the ideal time to be intervening. And it's very difficult to tell that these three beautiful children have an MPS condition. Many of them, the parent may bring them to the doctor with some degree of concern, but often, there are different sorts of treatments for the respiratory condition or some referral to occupational therapy, physiotherapy, speech-language therapy to help them with their delays. And it's not obvious that, in fact, these delays are due to something more serious. Next slide, please. And this one, Peter, if you could kindly just kind of tab through, shows this ... published. This is a boy who is- kind of tracked his progression of MPS2 over time with the citation in the corner to just see how difficult it is in the young years to appreciate what this condition is. Next slide, please. So now, I'm gonna spend some time talking about the outcomes with early diagnosis. So for Hurler syndrome, this is the neuronopathic form of MPS1. The approved treatment currently in the U.S. and in other parts of the world is hematopoietic cell transplantation, which helps provide the body with the capacity to make the missing enzyme. And because this is a progressive disease, we can actually see that there are differences in the cognitive trajectories of children who are treated earlier in their condition versus later. So, on the left, we can see that the trajectories of these individual children are much kind of tighter and closer around the kind of spread of the typical range of development. However, children who are treated after two years of age, we looked at another subset of the sample. You see there's a much wider spread and less likely to kind of hug the curve of typical growth. So in the U.S., Hurler syndrome, or MPS1, rather, is on newborn screening, and one of the reasons is because the difference in timing of treatment has really been shown time and again with the cognitive outcomes and certainly some of the other longevity and other functional outcomes. Next slide, please. MPS2 was more recently put on the Newborns Recommended Uniform Screening Panel in the U.S. for, again, the show of superior outcomes with timing of treatment. Now there is not presently an approved therapy to target specifically cognitive decline in the U.S. However, there is plenty of evidence from sibling studies and certainly from some work in Japan that there are superior outcomes for early treatment. In fact, what we have here is siblings who the younger brother had the benefit of early diagnosis in Hunter syndrome because it was known to test for it because of the effects on the older brother. And if we look at these two columns of symptoms, with patient one, this is the child who was diagnosed clinically versus patient two who was diagnosed because of his brother, we see that the left-hand column, the elder brother had many more clinical symptoms of the disease than the younger brother who was able to be treated early. Next slide, please. This is another symptom study that was done between two siblings. The sibling with the green arrow was the one who was diagnosed clinically and the sibling with the orange arrow on the bottom is the one who was diagnosed because of his older brother's diagnosis. On the next slide, I will be expanding the timeline. Go to the next slide. And it's still rather busy in terms of the different features, but what we see is that if we just kind of squint, the orange arrows are shifted more to the right, so there is kind of later onset of some of the clinical complications. And then I'm going to highlight some of those complications. Next slide, please. And what we see is that, in fact, even though they're slightly shifted to the right, we also see that there's less severe expression of the complications in the brother who was able to be treated earlier. So in this, the brother who was older and treated later became minimally verbal at age six. However, the younger brother who was treated at just beginning at 13 months of age was still speaking at seven years old at a five-year-old level. And remember, this is not a specific brain-targeted therapy. This is a peripheral enzyme replacement therapy for these two boys, and yet we're still seeing this benefit. Next slide, please. We also see differences in the severity of behaviors. So neuronopathic MPS2, as part of the cognitive decline, recall I was talking about all ways a person functions, has behavioral challenges as well. There's a pretty significant behavioral profile. And what we see is that the sibling who was treated when he was older had many more challenging behaviors that put him at risk. He had significant elopement. He was engaging in a lot of biting behaviors. His brother, who was younger, still had a behavioral profile of challenges. However, his elopement was just occasional. He was hyperactive, but he did not engage in these biting and pushing behaviors that were so challenging for the older brother. Next slide, please. Here are photos of the two brothers, again, comparing their joint disease. So the older brother, who had been receiving enzyme replacement for just six months, we see on the left, he's got the joint contractures. He also had difficulty even to raise his hand kind of above the brow line. And we can see the foot effects as well, whereas the younger brother, who was treated much earlier, when he'd received ERT for six months straight, we can see he's able to flatten his hands. He's able to reach above his head, and his foot is very different. But what if they have the benefit of 10 full years of enzyme replacement therapy? Next slide, please. What we see, unfortunately, is that what mattered the most for their outcomes was how early they were treated. So even though the older brother received ERT for 10 years, he was still not able to raise his head much above his- sorry, raise his arm much above his brow line. He still had significant joint contractures, and you can see how his foot is affected. Whereas the brother who was treated younger, the right-most column, you see he's much more able to flatten his hands, able to reach above his head, and his foot is not as affected. Next slide, please. So to summarize, treatment focus really must expand the concept of function to be really meaningful. There are many types of ways that the disease can affect individuals, not just with their cognitive abilities, but with their behaviors and their ability to interact with their environment. Community and family voices are critical. They're very critical to informing targets, thinking about the different ways that the diseases affect not just the individual, but also the families who are working very hard to take care of them. Next slide, please. We also wanna make sure to note that function is affected by many factors that are beyond these direct brain effects. These early symptoms, they're subtle, and they're challenging to detect. Presymptomatic intervention is really, really important, and earliest intervention, even if there are some of these early signs, which I hope these kinds of conversations will help illuminate, the earliest signs are the time to be treating and providing support. Even if therapies are not supposedly reaching the brain, there are still so many opportunities to improve quality of life and the whole person and the whole family. Next slide, please. There are so many people that I am indebted to for learning and collaboration, and I wish to express my thanks to them as well. So thank you. I think this time we'll move on to more panel discussion.

[00:49:15] Professor Roberto Giugliani: Yeah, great. It was great, Julie, really a great presentation and with very interesting data. I have a question to you about we see these guidelines that say that you should make hematopoietic stem cell transplantation in MPS1 before 24 months of age. Some countries, it's before two years and a half, and other countries like Brazil, it's three years, the guidelines say. What do you think is the real- the window that is reliable window to perform this transplantation and have a good outcome?

[00:49:55] Dr. Julie Eisengart: I really appreciate that question, and thank you so much for asking it. I think if I were only focused on cognitive outcomes, I would actually say the youngest possible, but I also want to appreciate that there is a lot more to a person than their cognitive ability. And when I think about things like transplantation, I think about the other incredible benefits of it. I also think about the other incredible benefits of enzyme replacement therapy. And I think about children's ability to exist with reduced pain, to move about and enjoy their lives, to have fewer respiratory issues and better sleep and better hearing and fewer cardiac concerns. So when I think about timing, it's easy to just say the earliest possible. And I think that even when families may not have the ability to pursue transplant so young, in Minnesota, where we have the privilege of newborn screening, we can do it at four months old, or at least my wonderful BMT colleagues can do it at four months of age. But knowing that that's really not a universal privilege, I think that it's fair to say that everyone who is able to access it, it's not useless to do it later. But I would still recommend as early as feasible for anyone, so under two if possible.

[00:51:45] Professor Roberto Giugliani: Thank you. Very, very, very clear.

[00:51:49] Dr. Julie Eisengart: And it looks like we might have time for a few questions and answers, if they might be submitted. Perhaps if we are waiting for- oh, here we see. Let me read this out. It says, "You've discussed hearing loss and MPS2. What percent of cases of MPS do you see ear infections and hearing impairment as one of the signs?" I might leave that one to you, Roberto.

[00:52:36] Professor Roberto Giugliani: Oh yes, very good question. In MPS2, almost all cases have hearing impairment. In other MPSs, maybe not that much, but I would say that if you investigate properly, for instance in MPS1 and MPS6, you will find that in most cases there's hearing impairment. And less frequent on MPS3 and MPS4, but in MPS7 also a very high degree of- a very high proportion of hearing loss. So it's, I would say, something that you need to pay attention and investigate because sometimes it's not clear and may impact the performance of the affected patient, the fact that is not hearing well.

[00:53:34] Dr. Julie Eisengart: Thank you, and I might add to that. I've heard from the community and certainly in the literature that many of the children with MPS2, sort of as they progress, may lose their ability to speak before they lose their ability to understand and that this difference can create some frustration. So I would imagine that there's some degree of link with the hearing impairment as well to that. I see we've received another question. It says, "Is a three-year delay in diagnosis typical? What can be done to decrease the delay? What can we learn from newborn screening in the U.S.?"

[00:54:26] Professor Roberto Giugliani: Well, I think that many things we can do to decrease the delay. One is to increase awareness of the community, especially health professionals, about mucopolysaccharidosis. And this is one part, but the other part is making the testing available, easily available. So this is something that some countries, some regions, it's difficult to have access to this test. And this is something you should work that also have the suspicion you can easily make the test and then you can reach a diagnosis and then can have access to treatment.

[00:55:13] Dr. Julie Eisengart: "Are upper airway infections more prevalent in certain types of MPS?"

[00:55:19] Professor Roberto Giugliani: Well, the MPS that have more somatic involvement as the MPS1, MPS2, MPS7, MPS6, they have more frequency of these respiratory infections. In case of MPS3, that is the involvement of the respiratory practice more attenuated, it's not so frequent. And also, but all these types of MPS, they are atherogenes, always have the severe and the more attenuated cases, and so they tend to be more concentrated in the severe part of the spectrum.

[00:56:05] Dr. Julie Eisengart: Okay, well, and I also do have to say that I've heard about- just kind of getting back to this question of a diagnostic delay, I've also heard that the community is incredibly strong, the worldwide MPS community, and they're incredible partners for research, for science, for medicine, and incredibly brave and generous. And I find that they are also working really hard to raise awareness within the community, reaching out, social media, working very hard to kind of educate and help in this effort. And they were enormous, enormous influences on newborn screening. It looks like there are no further questions. I'd like to thank the audience so much for your time and your interest. And thank you so much for sharing the stage with me, Professor Giuliani. Thank you for the opportunity to be here. And thank you all.

[00:57:08] Professor Roberto Giugliani: Thank you, Julie. You are an excellent chairperson. Very pleased to be with you in this session.

[00:57:15] Dr. Julie Eisengart: Thank you.