00:44:40
Transcription
[00:00:04] Prof. Roberto Giugliani: Hello, my name is Roberto Giuliani. I am a medical geneticist. I'm professor of medical genetics at the Federal University of Rio Grande do Sul in Porto Alegre, the very south of Brazil, and I work with lysosomal disease in a reference center for lysosomal disease, which covers a large part of Brazil, especially in the diagnostic point of view, and it's a pleasure to be here on the last session, last webinar of this series, on avoiding delays in diagnosis of MPS in children, unraveling the mystery of MPS clue by clue. So the title of my talk is MPS case studies, practical examples of an effective identification referral process. So I will discuss with you some ideas I have about how to improve the diagnosis on MPS and have some clinical cases to present and discuss. So I will present around half an hour, and then we'll have time for questions. The questions, I will show you, the questions should be posted in the chat, and then I will read the questions and I post the answers. So please do not take any screenshots and do not reproduce any of the slides, content, or images without express written permission of the speaker. So I will talk about half an hour and I'll have a Q&A session via text via the chat, and I will be very pleased to address your questions. So let's move to my disclosures. I have been involved in several, especially clinical trials on the MPS field.
[00:02:00] Prof. Roberto Giugliani: So the outline of my presentation, I'll talk about clinical suspicion of MPS, the importance of the early diagnosis, the evaluation of urinary GAGs as a diagnostic tool, the enzyme assays we can perform, the molecular genetic analysis, and some diagnostic flowcharts, and we'll end with a case report. So starting with clinical suspicion of MPS. So when should you consider MPS? Well, we should consider in a child that is born normal with slowly progressive curves. There are exceptions to this. In a small number of child, they may have hydrops fetalis and this may raise the possibility of MPS when you have this condition. But usually they are born normal and the curves are slowly progressive. Most patients present coarse facies. Not always. There are some types of MPS that do not have this. Corneal clouding is also suggestive but is not always present. For instance, MPS II, the Hunter syndrome, is not frequent to have corneal clouding. Hepatosplenomegaly is common to most MPS, but in some cases, especially MPS III, MPS IV, may be mild or not apparent. Joint stiffness is also dominant in most types of MPS, although, for instance, MPS IV may have joint laxity instead of joint stiffness. Bone dysplasia is also common in MPS, may be very important in some types, like molecule, like MPS IV, but may be mild in other types, like some Sanfilippo, MPS III. Also, you have frequently respiratory infections, respiratory distress. You may have heart valve disease, may be severe in some patients. It's common that patients have the report of previous surgery and especially hernia tonsils, adenoids, and some types of MPS will have cognitive decline. That may be the main characteristic in some types, like some Sanfilippo disease, MPS II. So, these are some ideas about when should you consider MPS. Now, I will show three patients and you try to figure out which patient has MPS. The patient number one in the left, the patient number two in the center, or the patient number three in the right. Or, maybe none of the patients or all of the patients.
[00:04:41] Prof. Roberto Giugliani: So, think about what you would say to my question, would reply to my question, and I will put the answer that in fact, none of these patients have MPS. So, they are, this day, the one in the left you see joint stiffness, but it has another condition called mucolipidosis. The patient in the center has hepatosplenomegaly, but he has another condition called Niemann- Pick B disease, and the patient in the right has a coarse facies, but has another condition called alpha-mannosidosis. So, the main conclusion is that you need the laboratory to make a diagnosis, even with a reasonable clinical suspicion. Let's go to a second question. Again, three phases. Which of these patients has MPS? So, in the left, in the center, or in the right, or none of them, or all of them? So, think about what you would reply to my question, and I will say the correct answer that is all these patients. So, all these patients have MPS. They have different types of MPS, but all of them have MPS. So, my first take- home message to you is that clinical suspicion is important, but you need the lab to make a diagnosis of MPS. So, let's move now to talking about the importance of early diagnosis, and my question is that easy to suspect MPS? For sure, it will be easy on this 11- year- old patient, but the same patient at six years of age, it's not that clear that he has MPS, and if you evaluate the patient at two years of age, you'll say this is a normal child. So, depending on the progress of the patient, you'll see this is how easy it may be to suspect MPS, but usually, you see young, and it's important to diagnose young, and at this point, it's not that easy to suspect MPS, but early diagnosis is important. I will show just one example from this paper published in the literature on a couple of two sibs. One sister diagnosed at three years and a half, and her younger brother diagnosed at eight weeks of life, and you see this. So, they were evaluated at three years and a half. The older sib, the girl, when she was just diagnosed, and the boy, three years and a half after having enzyme replacement therapy, and you see the facets of the two are completely different. You see the coarse faces on the left on the girl, and do not see this in the boy, but when you look at the spine, let's see the spine of the girl on the left, you see the scoliosis is very marked, and the boy is quite good shape, this spine, and you see the photos, the same thing. So, the difference, they have the same genotype, the same enzyme deficiency, but the difference is that the girl wasn't treated until three years and a half, and the boy was treated almost since birth. So, early treatment really makes a difference. So, my second take- home message is that efforts should be made to diagnose the MPS patient early in life. How to diagnose? Let's think, let's see the evaluation of the urinary GAGs, and if MPS is suspected, request a urinary GAG assay. This is very important and very informative, and I show in this picture, I'll take my laser pointer here, so that you see these orange dots are patients with MPS, and you see that they are all above the upper normal level, which is this green line here, and but one important thing that you also see is that there is a decrease, decreasing trend with age. So, it's very important to have age- related reference values, and also I would say to avoid diluted urine specimens that may lead to misleading results, and avoid also qualitative screening tests like this, the toluidine blue spot test, or the CTMA turbidity test. They can provide false positives. That's not a major problem, because we'll make some tests to clarify them, but can give false negatives, and this is a problem because you would miss this patient. So, avoid this, do the quantitative measurement of urinary GAGs, and always use age- related reference values. I have another question to you. When increased gag levels are found in urine, five options. These results confirm that the patient has MPS. Second, MPS suspicion becomes stronger, but the confirmation is still needed. Third, the information is almost useless due to the high proportion of false positives and false negatives. Four, none above. Five, all of the above. So, think 10 seconds on these five options, and I will show the answer. So the answer is that the MPS suspicion becomes stronger but the confirmation is still needed. So the GAGs are screening. It's a screening test for mucopolysaccharidosis, the urinary GAGs. You should move ahead but it's not useless. It's important because all types, almost all patients with MPS have increased urinary GAGs but it's not confirmatory. You need to move ahead. So be aware that some factors could interfere with measurements of urinary GAGs, some drugs, proteinuria and the contamination.
[00:10:50] Prof. Roberto Giugliani: So it's very important that the urine is transported and refrigerated and this may be a challenge especially for developing countries to have a refrigerated sample of urine being shipped to the laboratory. Also you may have some false positives and there are some diseases that have also increased urinary GAGs as mucolipidosis, as multiple sulfatase deficiency, as glycoproteinosis. So it's not specific for MPS. And there are also some false negatives that you may have with this test. GAG excretion decreased age.
[00:11:27] Prof. Roberto Giugliani: So when you have a patient that is older, maybe have a normal or near normal results. Also some types of MPS may have normal, near normal results like in MPS III and MPS IV. Some attenuated types may have this. So always be careful with this. And also with very diluted urine especially. And also that there is a variation of special urinary GAGs along the day. So if you have a normal result in a patient that you suspect, a very strong suspicion, maybe you can repeat in another sample to have a second result.
[00:12:05] Prof. Roberto Giugliani: So my third take- home message is increased total urinary GAGs suggest MPS but does not provide a diagnosis. And the normal levels of total urinary GAGs do not completely rule out a disease in a patient with a strong clinical suspicion of MPS. But they are very suggestive of MPS. And then we have a patient with increased urinary GAGs and now we can try to define one of the main phenotypes. One phenotype is what I call the visceral and skeletal phenotype. May have CNS involvement. And this occurs in MPS I, MPS II, MPS VI and MPS VII. The second one is a mainly neurodegenerative phenotype and this occurs in MPS III. One of the four subtypes of MPS III, A, B, C, or D. Or you can have a mainly skeletal dysplasia. That occurs frequently in the MPS IV, the Morquio syndrome, which may be the subtype A or B. So if you define one of these phenotypes, you can now have a bit closer to the diagnosis. One very important test that may help on the diagnostic process is the identification of the urinary GAGs species. Because there are several GAGs present in the urine. The heparan sulfate, dermatan sulfate, keratan sulfate.
[00:13:34] Prof. Roberto Giugliani: And so how they appear in the urine may be close to the diagnosis. For instance, dermatan sulfate occurs only, isolated dermatan sulfate you see only in MPS VI. Isolated heparan sulfate you see only in MPS IIIA, IIIB, IIIC, or IIID. Isolated keratan sulfate you see only in MPS IVA or IVB. And heparan sulfate, together with dermatan sulfate, you see in MPS I, II, or VII. So according to this pattern, you can focus your diagnosis. And the next step for the diagnosis will be the enzyme assay. So in a take-home message, in a patient with increased excretion, an abnormal pattern of urinary GAGs, and a suggestive clinical picture, diagnosis of MPS is very likely, but the confirmation is still required. So you have a clinical picture, you have increased GAGs, you have likely you have MPS, but you still need to confirm. And the way to confirm this usually is by enzyme assays. And it's important, too, because as I showed before, you see all these faces. They are patients with MPS, but with different types of MPS. And it's important to identify the correct type, because this is essential to provide a correct treatment, and also for prognosis and genetic counseling and prevention of future cases.
[00:15:03] Prof. Roberto Giugliani: So there are at least 11 types of MPS, maybe 12. There is a recent, recently was described another type of MPS that is the deficiency of arylsulfatase K, that may be MPS X, but so far we have these types of MPS, all the enzyme deficients you know. So, but knowing the enzyme deficiency, you know exactly which type of MPS you have. And this is because the there is a degradation pathway of urinary GAGs, and so if you remove the sulfate by the iduronate sulfatase, you then go progress in the degradation, you remove this bridge and you, by the alpha-L-iduronidase, then you progress in the degradation. But any enzyme that is missing, is deficient, you stop the degradation, you have storage of GAGs.
[00:15:55] Prof. Roberto Giugliani: So this is a very important that all these enzymes work, any of the enzyme that's not working, you have a interruption of the GAG degradation and you have accumulation of GAGs and in GAG tissues and gagging tissues and you have storage and you have increased urinary excretion. For doing the enzyme assay, in the past we use it to do in skin biopsies with growing fibroblasts. Fortunately, the techniques are developed to do this in blood and mainly leukocytes, sometimes in plasma, but mainly leukocytes.
[00:16:31] Prof. Roberto Giugliani: But now we can do most of these tests in dried blood spots, which is very convenient. In fact, the MPS investigation in dried blood spots is a practical sample for the assay of MPS enzymes. It is of interest to countries with some difficult to assess areas, as we have here in Brazil, where regular sampling and mail are limited. And also, DBS is easy to ship across countries and this may be needed sometimes. But assays in DBS are still taken as a screening, so patients which show positive results in DBS should be confirmed by additional biochemical or genetic analysis, maybe enzyme assays in leukocytes or maybe a gene analysis in the DBS. To assay the enzyme activity, the most common is to use artificial substrate, which this portion that is not fluorescent when the substrate is integral, but when you break the bridge with the enzyme, you have a fluorescence that you can measure. So you take the artificial substrate, you incubate with your sample, where you have the enzyme, you are testing, and if the enzyme is present, you have the fluorescent part is released, and you go to the fluorometer, and you measure the fluorescence, and you see that the controls have high fluorescence, and patients will have very low, maybe zero fluorescence. So this is a practical way to measure the enzyme. So you can change the artificial substrate, and then you change the type of enzyme you are measuring. It's really very convenient. I have to say that the MPS are usually heterogeneous, so even if you have one type of MPS, MPS II, MPS I, then there is a spectrum of severity. So in the case of MPS II, you have the more severe end, what we call the neuronopathic MPS II and you also have the more attenuated end, the non- neuronopathic , and some cases that fall in between, but usually there is a line, that is the cognitive decline, that differentiates the non- neuronopathic and the neuronopathic, the more severe and the more attenuated. But however, all patients have very low enzyme activity, so enzyme activity is not a way to differentiate between the severe and attenuated phenotypes. So the fifth topic, home message, all MPS patients have very low enzyme activity, irrespective of having the severe or attenuated forms. Also if low activity of a sulfatase is detected, some of the MPS enzymes are sulfatase. So we need to measure another sulfatase because there is a different condition called multiple sulfatase deficiency that is not an MPS but you have a deficiency of sulfatases. But you have deficiency of all sulfatases and this is a different disease. So when you have a sulfatase like in MPS II, it's an iduronate sulfatase deficiency. If you have a deficiency of an iduronate sulfatase, please measure another sulfatase to see if there's not the multiple sulfatase deficiency.
[00:20:13] Prof. Roberto Giugliani: It's very important. The fact is that when you measure the enzyme, you see very high levels in the controls and you see very low levels in the patient. So there is no any problem in differentiating these groups. But in carriers, this may not be the case. You have overlap between patients and controls. So for the carrier detection, usually you need to go to the molecular genetic analysis to identify this patient. So 6th take- home message, enzyme assay is useful for patient diagnosis but it's not reliable for identifying carriers. For this, you need the molecular genetic analysis. Fortunately, you know all the MPS genes. We know very well where they are, how many axons they have, what's the sequence. So we can sequence the genes and find the mutations. In the case of MPS II, we sequence the IDS gene and you find a mutation, you know what the patient is carrying. So in most cases, this is the 7th take- home message, a molecular genetic analysis should be performed to identify carriers.
[00:21:33] Prof. Roberto Giugliani: If the mutation in the index case is known, the lab should be informed to perform mutation-targeted genetic investigation, saving time and cost. So if you have a family, a case that you already know the mutation, you can say this to the lab and the lab will make a test for that specific mutation. This will be easier, quicker, and less expensive. And it's important to have the mutations because there is, in many conditions, there is a correlation between genotype and phenotypes.
[00:22:06] Prof. Roberto Giugliani: For instance, again, in the case of MPS II, we have a neuronopathic phenotype, patients more severe than non-neuronopathic patients with more attenuated disease. Usually when you have major rearrangements in the gene inversions, non-sense mutations, you have the more severe disease. When you have missense mutations, especially the ones that don't interfere that much in the protein, you have more attenuated disease. So it's important to know the mutation to predict or try to predict the phenotype, in addition to being important to detect carriers, to provide the genetic counseling, to make easier the prenatal diagnosis, and so on.
[00:22:50] Prof. Roberto Giugliani: So the eight take- home messages, molecular genetic analysis, may be useful for phenotype prediction and also for prenatal diagnosis and carrier detection. Let's go to the fourth question. Which of these statements about MPS is correct? Urinary GAGs are increased in both patients and carriers. Two, very deficient enzyme activity is observed in both patients and carriers. Three, genetic analysis allows for the identification of carriers. Four, MPS diagnosis is only possible by genetic analysis. Five, the only way to identify carriers is by genetic analysis. Oh, I will give you some time, I already showed the correct answer, but let's let's discuss.
[00:23:45] Prof. Roberto Giugliani: So the correct is three, genetic analysis allows for the identification of carriers. Why one is not correct? Because urinary GAGs are not increased in carriers, only in patients. Also, the second one is not correct because very deficient enzyme activity is observed only in patients, not in carriers. Carriers usually have some degree of enzyme activity, although it may be below the normal level, but maybe usually is not very deficient enzyme activity. Four, it's not common because MPS diagnosis is possible by biochemical assay, by enzyme assay, not only by genetic analysis. And fifth is not common because it's not the only way to identify carriers by genetic analysis, because you can identify carriers by pedigree, by analysis of the family. If you have an MPS II family, the X- linked MPS, and you go to the pedigree, you can make the diagnosis of carriers and you can identify carriers by the family tree, so it's not only by genetic analysis. So because of this, the third option is the correct.
[00:25:01] Prof. Roberto Giugliani: So let's go to the flowchart. What's the usual diagnostic process? You start from the clinical suspicion, you go to the GAG analysis, quantitation of GAG, identification of GAG species, then you go to the enzyme assay, and you have already at this point you have a diagnosis, but usually you go to a molecular diagnosis, confirm the mutations, and you have the process completed. What is becoming more and more common now is you have a clinical suspicion, and then you go to the molecular analysis. So you do the targeted mutation detection, or gene sequencing, or some gene panels, or even whole exome. And so the molecular genetic analysis is becoming more and more the second step. First you suspect by clinical suspicion, and then you go to molecular analysis, because you have a panel that is easy to perform, much easier than an enzyme assay for some of you. But even if you have the molecular diagnosis or molecules, you need to go to do the enzyme assay to confirm that you have these molecular variants, these genetic variants you find found in the genotyping. They have an impact on the protein, so we need to do the enzyme assays, and also that the enzyme assay that shows deficiency has impact, and it leads to gag accumulation. So you need to do biochemistry to confirm the molecular diagnosis as is usually you go to the molecular analysis to confirm the biochemical. So either way you do the testing is okay, but you should have the complete picture at the end. But there are several progresses on this, and now you have some tests that even performed in urine, they are not the measurement of total GAGs, but the specific signatures you find in urine when they analyze by tandem mass spectrometry, that are very specific for each type of MPS. For instance, you see an MPS on the MPS I, the top, then the MPS II, the MPS VI, the MPS VII, all different patterns, and you can suspect of the diagnosis with this kind of signature, and this is becoming more and more frequent. So there are several ways to look for patients with MPS. One is departing from the clinical suspicion, usually using a biochemical screening. We can also screen high- risk groups, like patients with short stature, patients with joint restrictions, and then you use biomarkers like GAGs or gene panels to screen these patients, and you have more patients detected, but you'll see not all patients will be detected. You'll miss some patients in the population.
[00:27:59] Prof. Roberto Giugliani: To detect all patients in the population, you need to do mass screening, and this may be, newborn screening may be the way to approach this, maybe starting from biochemical approach or for genetic approach. So this is already, the U. S. has already recommended the MPS I, the screening for MPS I, and now it's also for MPS II, and this, I think, will become standard, and not only for the U. S., but for the many countries in the world in the coming years. So now I will finish this webinar with a case presentation to illustrate some of the things I mentioned. So it's a patient from Brazil, BOC, is a patient born from normal, non- consanguineous parents with no further cases in the family, normal physical and psychomotor development, had adenoidectomy, tonsillectomy, and surgery for inguinal hernia in childhood. When he was six years old, he had restrictions in joint mobility that were noticed. He performed an echocardiography at the age of seven years, showing the mild thickening of mitral and aortic valves with no systemic repercussion. After investigation by several specialists, MPS was suspected, investigation was requested to our diagnostic network, that is the MPS Brazil network. So how it works, the MPS Brazil network, so have a family that has a patient who suspected MPS, then they go to the doctor, the doctor collects samples and send to the headquarters of the MPS Brazil network located in my city, in my hospital in Porto Alegre. We receive every day many, many samples from all around the country. We process these samples and we provide the results directly to the doctor and the doctor provides it to the family. We raise money from several sponsors to maintain this program in order to provide these tests at no charge to the family or to the doctor. Usually we start by measuring the urinary GAGs and in this patient we found an increased level of urinary GAGs. Then we go to look at the species of GAGs in the urine and in this patient we found heparan, dermatan sulfate pointing to MPS I, MPS II, or MPS VII. And then we did the enzyme assays and the enzyme assay for iduronate sulfatase. When you couple the artificial substrate with the patient's sample, we had this artificial substrate fluorescence measured by fluorimetry and you see this patient's very low activity confirming the diagnosis. Then we went to the genetic analysis of the IDS gene and we found this mutation in the IDS gene further confirming the diagnosis. So the laboratory investigation showed the urinary GAGs increased 495, the upper limit was 106 for the age, the electrophoresis of urinary GAGs showing heparan and dermatan sulfate compatible with MPS II, iduronate sulfatase was 3. 3, the normal was 122 to 463. And as it is the sulfatase, we need to measure another sulfatase and we measure arylsulfatase B that was normal, within normal range, so ruling out multiple sulfatase deficiency. And we found a mutation in the IDS gene that further confirmed the diagnosis. This was a pathogenic mutation already described in MPS II patients. We discovered that the mother also carries the mutation, can provide genetic counseling to this family and also to other members of the family. And we saw this patient was diagnosed very late, 13 year old. And this is very common, at least in Brazil, but it's common worldwide. And we made a study looking at the history of over 100 MPS patients and what happens from the birth to the biochemical diagnosis. And we found that the symptoms, they started around, on average, before 24 months of age, but the diagnosis takes almost 5 years, 4.8 years on average, to be reached. So it's a long delay, especially when they have specific therapies available that provide a better prognosis to this patient. So it's very important to shorten this diagnostic journey of MPS. And one thing you know, we were observing in this study, that most patients, 6- 7 on 113, so they already had performed a surgery, like hernia, adenoidectomy, tonsillectomy, ear tubes placement, or other surgeries. And so they went, they were admitted to a hospital, they had a surgery, and nobody suspected of MPS. So it's about 60% of the patients. So this is very important to talk about this. So the patient started a year, he was diagnosed at 13 year old, and this was certainly good for him, because he can improve the respiratory parameters, the sleep pattern, the walking test, the liver volume, the urinary GAGs, auditory tests as well. There are things that do not change with ERT, so there's still a medical need, echocardiography, and visual acuity, and the patients with cognitive decline is not a case of this patient. Also the enzyme replacement therapy did not improve the cognition, because the enzyme does not cross the blood-brain barrier. So he's the patient after the therapy. And how is it will change the diagnostic laboratory for MPS in the future? I think we'll have a laboratory with just two equipments, a tandem mass spectrometer, where we can perform the measurement of GAGs, these individual GAGs and also can perform the enzyme assays. And the sequencer where you can do the, you'll find the mutations in all the MPS genes, and all of these you can do in dried blood spots. So this will be very much simpler than in the future. So summarizing... If you suspect an MPS measure, urinary GAGs. If GAGs are normal, think of non-MPS lysosomal disease . But if GAGs are increased, further tests should be performed. Identification of gagged species together with clinical and radiological findings could suggest potential diagnosis. Detection of enzyme deficiency is recommended for the diagnosis. And molecular genetic analysis is useful for diagnosis confirmation, carrier identification, and prenatal diagnosis. If molecular genetic analysis is performed before biochemical tests, enzyme assays and urinary GAGs are still needed to confirm the diagnosis. And identification of the correct MPS type enables therapeutic decisions and correct guidance to the family. So now, thank you very much for your assistance. Now it's time for the questions and answers. And I am open to your questions. I will remove my laser pointer and I will bring this shot. So you can use the shot and we can have some questions. And I'm open to that. Let's see. Okay.
[00:36:23] Prof. Roberto Giugliani: So I have already a question. That is, is clinical suspicion straight to molecular analysis better than traditional root urinary GAGs and enzyme assays or not? Which one is quicker to start treatment? Well, I would say that both ways of doing the diagnosis are okay, but you should do the complete set of tests. So if you go from clinical suspicion to molecular analysis, you still need to confirm the enzyme deficiency and the storage of GAGs.
[00:37:03] Prof. Roberto Giugliani: If you go to clinical suspicion to storage of GAGs and enzyme assays, it's recommended to do the molecular analysis to have the full picture. So I would say that you start treatment when you have the full picture. So I think there is not a shorter or quicker way to do this. It may depend on what you have available, but I would say that the full picture is still important. In some cases that we cannot have access to all the tests, we may do, for instance, if you have a clinical picture, you have increased GAGs, and you have pathogenic mutations, okay, you can have the diagnosis. If you have the clinical suspicion, urinary gags, and the enzyme deficiency, you also have a confirmation. But then they want to be quicker depending on the particularities of each one, and easier access to the molecular genetics or easier access to myochemical testing depends on the situation.
[00:38:19] Prof. Roberto Giugliani: But it's important to have the situation well confirmed to start therapy, especially because therapy is very specific, and you need to know exactly which type of MPS you have. So there is a second question arriving here. Urinary GAG screening tests, false positive or negatives, how common are they? Very good question. It depends on the test used. As I said, you should avoid the qualitative or semi-quantitative tests like these spot tests in urine, like these turbidity tests. You should go to the measurement of urinary GAGs at the screening test.
[00:39:07] Prof. Roberto Giugliani: This is very powerful, I would say, that may have some false positives. You can have false negatives if you have diluted urine specimens, or if you have a patient that is older, like the second decade of life, especially sometimes MPS III or MPS IV, they may have some near-normal values. So I would say that...
[00:39:34] Prof. Roberto Giugliani: when you have a normal result, when a patient that you have a strong clinical suspicion, do not give up of your idea of having MPS. Do a second test or do a more... go ahead in the investigation doing the observation of the GAG species or the enzyme assays or the molecular genetic analysis. So it's possible to have false negatives. They are not common, but it's possible. False positives are more common, but I'm not worried with false positives because when you progress in the investigation, you clarify that.
[00:40:14] Prof. Roberto Giugliani: But always take into consideration the clinical picture and the interpretation of your results. There is another question. What do we have to do when you have a strong clinical suspicion of MPS with positive GAGs and normal enzyme activity? So this is a very good question. We have a suspicion, but you have a suspicion. GAGs are increased, but enzyme activity is normal. Well, I think that we should look for, there are 11, maybe 12 enzymes involved. So we need to check all these enzymes to see if they are really any of these enzymes is not increased. And even if you have but you can think of some conditions that may have increased GAGs, may have this clinical picture of MPS, increased GAGs, that are not MPS. I would say mucolipidosis is something that you will not find enzyme deficiency of the GAG breakdown pathway. Also the multiple sulfatase deficiency, but then you can have some enzymes involved. And there are some conditions that may have increased GAGs, but are similar to MPS, other lysosomal disease. So you should do not give up on the investigation. When you have a clinical picture and urinary GAGs, you should have an explanation, even if it's not an MPS. Wow, more questions. My question is about the geographic distribution of MPS. Is it commoner in any particular races or regions? So this is also a good question. There are some MPSs that, like in Brazil, have a proportionally high number of MPS VI, the Maroteaux-Lamy syndrome. It's about 23% of all our MPS cases. Very, very different than what we have in Europe or in North America. That is, the disproportion of MPS VI is very low. But you have in North America, the majority of MPS are MPS I, which is not the case as it is in Netherlands, that we have more MPS III. So there is a variation of distribution of MPS from country to country. So some are more common in the south, some are more common in the north. And so you have to, this is related to the genetic background of the population, the more prevalent mutations in this area. For instance, in Turkey is very common the MPS VI and also the MPS IIIB. And in other, in Japan is more common the MPS II. So there is a difference from region to region and you should consider this. But in the full set of MPS, there is a reasonably homogeneous incidence of around 1 to 21 to 25 thousand individuals is the average for all the MPS as a whole.
[00:44:06] Prof. Roberto Giugliani: Okay, so if you have no further questions, I want to thank you very much for your audience, not only for today but for all this session on MPS. It's very important that we talk about MPS and we discuss the ways to make the diagnosis and enable these patients to have an earlier diagnosis and an appropriate therapy whenever possible. Thank you very much.