Prof. Maurizio Scarpa: 00:00:00 Thank you very much. This is for being here. I mean, this is Maurizio Scarpa, and I'm speaking from Orlando, in Florida, attending the World Meeting 2020 on Lysosomal Disorders. I'm Maurizio Scarpa. I'm a pediatrician. I work... I'm working on Lysosomal Disorders since 30 years. A long time. And today, what I would like to show you and discuss with you is the challenges in diagnosis and disease management of MPS and alpha-mannnosidosis.
Prof. Maurizio Scarpa: 00:00:50 In this webinar, which is part of a series of webinars regarding MPS and alpha-mannnosidosis, and discussing different aspects of these two Disorderss. So, Orlando is a very nice city. This is a meeting run by Chet Whitley, and it's the 16th edition. It's a meeting attended by about 2,000 people, and the meeting is touching all the different aspects of pathophysiology, and clinical, and new therapies. new things that have been said in these first two days. So, as I said, the title is MPS and alpha-mannnosidosis challenges in Disease Management in Neurology.
Prof. Maurizio Scarpa: 00:01:49 Just a couple of indications before starting. You should not take any screenshot. Please don't reproduce any slide. I will try to be with you for 45 minutes. You can submit your question at any point in the discussion. And then please submit via text and not verbally. And if not possible to answer, please record at the www. ineip. org. So, the presentation we touched, the first one is to understand the pathogenesis
Prof. Maurizio Scarpa: 00:02:38 genes,
Prof. Maurizio Scarpa: 00:02:38 We have about 23, 000 genes and more than 13, 000 are expressed in the human brain. We have about 1, 200 different inborns of metabolism. And more than 400 actually involve the central nervous system. The interesting thing is that we have also specific genes. 33% of them are actually expressed only in the brain. And the brain diseases can be divided into major fields, one which is common diseases, and then others that are individually rare disease, but all together affecting 10% of the world populations. Rare diseases, you know everything about.
Prof. Maurizio Scarpa: 00:03:58 I mean, they are a group of 6,000 Disorders, about 10% of all the known human diseases. They are affecting five, less than five, than 10,000 people. And they are 80% genetically origin affecting about 3% to 6% of the European population. Lysosomal Disorders is a group of 70 diseases. Altogether, they have an estimated frequency of one every 5,000.
Prof. Maurizio Scarpa: 00:04:28 And according to the substrate that are accumulated, they can be divided in glycogenosis for glycogen, storage glycolipid, mucopolysaccharides, oligosaccharides, transplant enzyme deficit, and lysosomal membrane transport enzyme deficit. These two categories are new and they are enlarging the possibility of having defects of enzyme that are ending in a storage of a substrate.
Prof. Maurizio Scarpa: 00:04:56 The basic concept of neurological involvement was very simple, let's say 30 years ago, when it was said that the only reason to have a deficit is just because of a cytotoxicity induced by the substrate that is accumulated and intoxicating the cell and bringing the cell to death. One gene defect, one protein defect, one substrate.
Prof. Maurizio Scarpa: 00:05:25 state, the substrate is accumulating, the cell is suffering, and the cell then is going to die. At that time, it was the only thing that we could know due to the scarcity of knowledge about these processes. But what we saw then is that if you see the organs that are accumulating, in this case mucopolysaccharide like liver and spleen, you see that it is quite evident the accumulation over time.
Prof. Maurizio Scarpa: 00:05:56 But if you see the accumulation in the brain, these are slices of patient brains, you see that the accumulation is only in the minimal part, not inside the parenchyma, mostly in the coroidal plexus. And in the parenchyma, you see just some buckles due to the hypertrophic lysosome. The accumulation of the substrate is in all organs since the lysosome is in all cells of all organ , and 70% of patients have some CNS involvement. One thing that is linked to a CNS involvement is the accumulation of Heparan Sulphate.
Prof. Maurizio Scarpa: 00:06:44 If you see here, for example, in the MPSs, the Heparan Sulphate is concentrated and you have neurological problems. So there is a strict dependence by the concentration of the Heparan Sulphate and neurological effect, and indeed neurological involvement can be present in the severe form of MPS1, MPS2, in the severe form, in all the MPS3 that are indeed accumulating only the Heparan Sulphate. And the Heparan Sulphate is present also in MPS7. Why?
Prof. Maurizio Scarpa: 00:07:19 Because the Heparan Sulphate is responsible of a cycle of inflammation and the Heparan Sulphate, when it is accumulated, is generating a cascade of release of cytotoxin, TNF-alpha, and interleukins that are really producing a lot of inflammation and for this reason cell apoptosis. What is the effect of this?
Prof. Maurizio Scarpa: 00:07:49 The effect... of these patients, the neurons are actually atypical and in all the different parts of the brain, what they have is an ectopic dendritogenesis and inside these ectopic dendrocytes, there is... spheroid molecules rather than lysosomes, altered mitochondria that are altering the synaptic capacity and the classical formation.
Prof. Maurizio Scarpa: 00:08:20 So the proposed model of cell death in LSDs is that you have here an enzyme deficiency, you have indeed a substrate storage, but you start with accumulation of toxic protein and its function of mitochondria and then you have as a consequence cellular distress, activation of microglia, chronic inflammation and cytokine release and at the end the cell is actually going to go in apoptosis. The clinical features in MPSs and alpha-mannn. The clinical feature is related to all the different sign and symptoms in MPSs.
Prof. Maurizio Scarpa: 00:09:09 You have MRI abnormalities, problems of hyperactivity, sleep Disorders, cognitive problems, motor skill impairments, abnormal gait, swallowing difficulties, abnormal reflux, EEG alteration and seizures. The MPSs that are mostly affected are the MPS1, MPS2 type A, the MPS4, MPS3, the MPS7 and some, although there is very little knowledge about the MPS9. The differential diagnosis is of course with mucolipidosis, with the alpha-mannnosidosis and the multiple sulfatase deficiency.
Prof. Maurizio Scarpa: 00:09:46 As you see here, the phenotype of these patients are very similar to MPSs so I will treat these four diseases for this specific part of clinical phenotype as a unique group.
Prof. Maurizio Scarpa: 00:10:04 The neurological manifestation is affecting all the different ages of the patients. As you see here, in the very first five years, you have more presence of cognitive problem and behavioural problems, and hyperactivity but with growing of the patient you are adding abnormal gait rather than either cephalos or abnormal reflex. One important thing is that we will discuss later is rge presence of Carpal Tunnel Syndrome. The Carpal Tunnel Syndrome is specifically for MPS. It is not a pediatric disease, and whenever it is seen, a differential diagnosis with lysosomal Disorders should be taken into consideration. Here there is a schematic model of CNS involvement. You see that the phase of CNS involvement can take 15 years, and actually you have a pre- symptomatic phase which is lasting about one to two years.
Prof. Maurizio Scarpa: 00:11:10 Then the disease is starting with a language delay, behavioral problems, and then regarding about four to fifteen years deterioration of cognition and then motor skills, and this is actually coupled to other symptoms like hyperactivity, which is together with irritability, starting very early in the history of the disease, and then being more progressively higher and then when the disease is very much established and the situation is very compromised, the hyperactivity is decreasing.
Prof. Maurizio Scarpa: 00:11:49 As you see, there is a continuum of symptoms, and if we are very, very careful, we can recognize in the pre- symptomatic part also some symptoms that can give us a clue in order to make an early diagnosis.
Prof. Maurizio Scarpa: 00:12:09 The neurological features that we will go to discuss now, what I would like to show you is a little bit about the different aspects of MRI alteration that we are commonly seeing in these patients, and the neurological symptoms are actually cervical pain, hyperreflexya, weakness and motor defects, and when the situation is very severe, you have sphincteral disturbances, headache and vertigo, and then you have apnea and syncope.
Prof. Maurizio Scarpa: 00:12:47 The tests that we are using are all the tests usually performed in neurological analysis and of course electroencephalography and somatostasic potential together with the acoustic and the visual potential are, of course, major tests that are of great help for our diagnosis. The neuroimaging that we use are the X- rays of the skull, and we will see some, and then a patient needs to be evaluated also for the spine, because the spine is very much involved not only on an orthopedic but also on a neurological and then of course we have CT scan and MRI in order to understand the parenchyma alterations. Let's analyze a little bit this kind of alteration.
Prof. Maurizio Scarpa: 00:13:39 The first one that I want to discuss with you is the fact that it is typical of these patients to have enlarged perivascular Virchow-Robin spaces that you can recognize here as spots, and sometimes they are like bees' nests in the brain of these patients. These are due to infiltrated vascular spaces with the GAGs. They are not related to the severity of the CNS. They are present also on patients that do not have CNS alterations, but in any case they are an evidence of the alteration of the vascularization of the brain.
Prof. Maurizio Scarpa: 00:14:22 Another important sign is the white matter abnormalities, which is especially surrounding the ventriculi, and these are due to microglia activation and reparation. It's a sort of repair event. Another important abnormality you see in the brain is actually the atrophy.
Prof. Maurizio Scarpa: 00:14:52 The atrophy is according to the age of the patient, and, of course, when there is atrophy, there is enlargement of the CSF space and a substitution with CSF of the tissue that is lost, and you can have this kind of gigantic hydrocephalus and enlargement of the lateral ventricles in older patients.
Prof. Maurizio Scarpa: 00:15:20 The ventricle peritoneal shunt is a therapy when you have an increased endocrinic pressure, and this is something that is important to study because the endocrinic pressure is not very comfortable to diagnose in these patients because it is very slow, it is not sudden and it can be the cause of the irritability and sleep Disorderss. So the MRI is extremely important to do and whenever there is a suspect of an endocrinic pressure it is important to have a lumbar puncture in order to measure the pressure of these patients.
Prof. Maurizio Scarpa: 00:16:14 Of course, the ventricle peritoneal shunt is a procedure which is routinely done in endocrinic hypertension, but it is not free from complication, which can be early or late complication, and you can have infections, bleeding rather than bowel perforation and fistula, and in the late one you can have the dysfunction of the system. Sometimes the pump is actually not working properly and needs to be repaired.
Prof. Maurizio Scarpa: 00:16:49 An important note is that in non-MPS patients, hydrocephalus is symptomatic and the signs of intracranial hypertension are usually present: headaches, vomit, pressure alteration, papilledema. This is something that is very much helping normal children, but in MPS patients these symptoms may not be present because the hydrocephalus can be the result of increasing atrophy. The hydrocephalus can develop very slowly and the symptoms may be very attenuated.
Prof. Maurizio Scarpa: 00:17:22 The papilledema cannot be considered as an intracranial hypertension sign due to the fact that the papilledema is always there, which is not sometimes a real papilledema, but it is an infiltration of the papilla with drugs. The shunt should be considered when there is a real ICH and for knowing this a lumbar puncture is often recommended. Another brain abnormality which is very typical of these patients is the J-shape of the sella turcica. Usually the sella turcica is much more closer. Here you have an omega profile and here then you have the hypothesis.
Prof. Maurizio Scarpa: 00:18:12 This is a typical sign which is seen in most of the LSDs and in particular in MPS. Another important sign that you can recognize in the MRI is the hyperostosis of the calvarian bones that you see here, and indeed these bones are much thicker. This is also indicative of an MPS and alpha-mann possibility. Another sign that has been found specific for the MPS is the closed meningoencephalocele.
Prof. Maurizio Scarpa: 00:18:45 The closed meningoencephalocele is actually a protrusion of the brain that you can find in the anterior part of the brain or the posterior fossa, and it is due to the softness of the bone in the very beginning of the life of these patients, and the CSF and the brain are actually protruding.
Prof. Maurizio Scarpa: 00:19:12 This is a very specific sign that has not been found in any other disease so far, and it is responsible of herniation of the brain, which needs to be taken into consideration, especially when you are intubating the patient from the nose, because here there is the hyoid bone which is very, very fragile and you can break it due to the fact that this is even more fragile due to the herniation of the brain. And if you see the basis of the skull of these patients, you see that there is a sort of erosion due to this closed meningoencephalocele.
Prof. Maurizio Scarpa: 00:19:56 This is something that is very typical and when it is seen, the MPS diagnosis should be immediately searched for. Not only the brain and the skull are affected, but we have a lot of abnormalities in the spine. We have dural thickening, we have cervical stenosis, spinal cord myelopathy, atlanto- occipital assimilation, basilar invagination, dens dysplasia, vertebral abnormalities- all the different parts of the spine are actually affected. One important thing is the dural thickening and dens dysplasia.
Prof. Maurizio Scarpa: 00:20:34 This is due to the accumulation of the GAG in the spine. You have here accumulation and a compression, a cervical compression which is particularly present in MPS4, MPS6 and MPS1 and a little bit less in MPS2 and MPS3, but it becomes smaller and smaller and surgery is most of the time required.
Prof. Maurizio Scarpa: 00:21:00 As you can see here in the normal patient, the flow of the CSF is very much conserved. In the severe, you see that there is a compression of the cervical spine, which is also sometimes, most of the time, giving a myelopathy due to the fact that the CSF circulation is really affected. And you see here how it can be, from a normal, also the diameter of the cervical spine after compression. So at the end, what you can have also is an invagination, so that in certain conditions, the cervical spine can be even more compressed by the movement of the patient.
Prof. Maurizio Scarpa: 00:21:52 So for this reason, whenever we go to analyze this district, if you do an x-ray or if you do an MRI, you should do it in a flexo extension position in order to see whether there is an invagination and a restriction of the cervical spine due to the movement. The therapy of this is actually surgery. You have to open the canal and you have to make a cleaning of the position, and sometimes, especially in some very severe MPS4 patients, the destabilization of the column is such that you need to fix.
Prof. Maurizio Scarpa: 00:22:40 So this is something that is needed to be thought and done before the surgery itself. The main diagnosis in the craniocervical stenosis is, of course, the neural examination, and then the SEP of the middle nerve, and then the MRI. And all of these are altered, because the MRI we have seen, a biological examination, you can have a vivacity of the reflexes, and the SEPs are abnormal. In the additional one test, you can use pulmonary function, because sometimes
Prof. Maurizio Scarpa: 00:23:22 you have pulmonary restrictions.
Prof. Maurizio Scarpa: 00:23:33 And then you have a six-minute walking test, which is a direction of the CT scan. You can see the alteration due to the accumulation and the compression of the cervical spine. Of course, one thing that is important is also to measure the somatostasis potential of the tibial nerve, which are altered and is indicating a compression of the cervical spine. Another important problem that is specific of MPS and Alpha-mann patients is the carpal tunnel syndrome. The carpal tunnel syndrome is something due to the accumulation in the medium nerve of GAG, and creating a lot of bad problems of pain, numbness and pain in the first three fingers.
Prof. Maurizio Scarpa: 00:24:19 This is something that the children can have affected by MPS from the first two or three years of age, and for them it is not very abnormal. So they may not be telling about this discomfort as other patients, more adults. So these children actually survive with this pain and with this feeling for a long time, but it is something that is really very much painful and uncomfortable, and this can be also another important symptom that can cause hyperactivity and irritability.
Prof. Maurizio Scarpa: 00:25:01 You see that this is very much possible and present in these patients due to the fact that there is the accumulation and also the joint stiffness of the fingers, and the fact that you have this accumulation is giving sometimes the child a very much uncomfortable situation. So one thing that needs to be done in order to make diagnosis is to measure the nerve conduction velocity, which is altered as you see here, and it is done in a very simple way at all.
Prof. Maurizio Scarpa: 00:25:48 For the children sometimes it is a bit painful, but if you see this alteration then the only thing you can do is to make a surgery in both hands and clear the medium nerve in order to release it from the accumulation.
Prof. Maurizio Scarpa: 00:26:07 Whenever you find in a child a carpal tunnel syndrome the MPSs and the lysosomal Disorders need to be ruled out because it is, as I said, not a pediatric usual disease and it is very uncommon and, if present, most of the time, 90% of the time, are related to the MPSs. Not only these are the difficulties of these children with the CNS. I mean, as we said, we have, especially in the first couple of years, a lot of behavioural aspects that need to be taken into consideration.
Prof. Maurizio Scarpa: 00:26:49 Hyperactivity, hyperorality, the lack of fear, the risk for themselves, the agitation, the aggression and the abnormal social interaction, together with sleep disturbances, are major symptoms that can be seen in the first three years of life and, of course, are the ones that most of the time lead to a diagnosis of a severe phenotype. Here is a table, and I will invite you to see the webinar on the website, which is linking the development of the behavioural aspect with brain manifestation.
Prof. Maurizio Scarpa: 00:27:32 I think that this is a table that can be very useful in order to link the cognition in different MPSs and when the plateau starts to begin rather than the progressive deterioration. It is something that we should take into consideration in the analysis of the patients in the very first six to seven years of age. Sleep disturbances is one of the first symptoms in these patients. The most problems are due, indeed, to neurological problems. One important thing is that the accumulation is altering the expansion of the circadian genes.
Prof. Maurizio Scarpa: 00:28:18 We have documented that in MPSs and also on other diseases these circadian genes are actually giving the child a rhythm which is not normal. They are usually more sleepy in the afternoon rather than in the evening, and this can lead to a lot of distress in families. There is not much to do until we have an ERT or a therapy crossing the blood- brain barrier, because it has been seen that whenever the storage is actually clear, even the circadian genes are going back to normal.
Prof. Maurizio Scarpa: 00:28:55 By now, what we can do is actually to give some melatonin and different milligrams. Sometimes it is not working, not because the melatonin is not produced in the right concentration. Actually, the melatonin production is totally normal, but it is the rhythm of the melatonin production which is altered and leading to a sleep disturbance. Of course, this is one problem. Another problem in the severe CNS patients is seizures. This is particularly a symptom that is starting from the fourth to the fifteen years of age. Seizures occuring in 30% of the patients with MPSs.
Prof. Maurizio Scarpa: 00:29:40 MPS2 and MPS3 are more prone to develop seizures, and the problem is due to the alteration in the synapses communication. This has been shown very clearly in animal models and in biopsies of brain patients. The synaptogenesis is altered, and for this we may have an additional problem due to the storage accumulation. An interesting observation that we did is that there are some patients that are particularly hyperactive and particularly agitated overnight.
Prof. Maurizio Scarpa: 00:30:20 If you do, as everybody should do, a polysomnography with the EEG, there is a particular nocturnal frontal lobe epilepsy, which is actually activated or generated or starting with the sleeping which is present overnight and is giving an alternation of EEG that whenever regognised can be corrected and ameliorating the behaviour and the sleep in these children. These children are behaving see that the movement of the children are always the same, and whenever you see this rolling during night of this child this maybe worth this child is affected then with a polysomnography you can see this non- convulsive status and can be really very well treated.
Prof. Maurizio Scarpa: 00:31:17 Early markers for CNS diseases, there is a very nice paper some time ago by Maria Escolar recognising seven major clinical markers that are listed here and we have partially discussed the behavioural difficulties, the increased activity, the sleep disturbance, the seizures, together with the preservative chewing and the inability to bowel training and the inability to bladder training. These are other three symptoms that are present and whenever they are present in children over two years of age can be also a sign of potential MPS or alpha-mann.
Prof. Maurizio Scarpa: 00:32:01 How do we test the children for behaviour? There are different tests that are available worldwide and these tests are important to be given at the right time and with these tests you can follow the progression of the disease and you can even activate some additional therapies like logopedy and behavioural therapy to try to delay the onset of progression. And for all the patients you can have an analysis of the IQ rather than memory and adaptive function, rather than attention and executive function, and visual spatial ability and these are all tests that should be done routinely at least once a year or more often when the child is starting to progress. What about treatment? Treatment is actually with the CNS not very much effective.
Prof. Maurizio Scarpa: 00:33:22 I mean we don't have at the moment any kind of therapy to decrease the progression, to stop the progression of the disease. At the moment the only therapy that has been shown to be efficient in some patients to delay or even to change the natural history of CNS is bone marrow for the MPS1 hurler. Hematopoietic stem cell is indeed a gold standard therapy for children under one year of age with MPS1H and it has been shown that if done early it is decreasing or even changing and we have very good experience with this therapy but this is the only MPS in which bone marrow and HSCT is working.
Prof. Maurizio Scarpa: 00:34:29 Now we have in some countries a newborn screening for MPS1 and of course if the newborn screening, the genetic screening, the enzymatic analysis is actually predicting a hurler phenotype, the gold standard is to propose the HRT. There are experiences on MPS2 and MPS3 but actually there is less or no beneficial according to the groups that have performed this therapy regarding the effect.
Prof. Maurizio Scarpa: 00:35:07 For sure it is not like the MPS1H. At the moment there are some treatment options like the intrathecal enzyme replacement therapy, there are trials ongoing, the success is on the way of delivery, there is a presence of enzyme in the CNS, there is a facility in injecting enzyme, there is a decrease in GAG level, is very well tolerated but actually the effect is not as we would like, that means that the disease is progressing no matter this treatment.
Prof. Maurizio Scarpa: 00:35:49 Good sign. So we need to indeed work more on that. Other treatment option is therapies crossing the blood- brain barrier. Now we have a lot of different approaches. We have large molecules that are fused with receptor linking to the blood- brain barrier. We have nanoparticles that are loaded with enzymes that can cross the blood- brain barrier due to the presence of linkers for the blood- brain barrier.
Prof. Maurizio Scarpa: 00:36:28 Small molecules can passively cross the blood- brain barrier and try to reduce the substrate or to increase the capability of processing the enzyme that sometimes for genetic reason is entrapped in the maturation of the enzyme to the lysosome. And there are interesting clinical trials for this, but at the moment there is still need of asserting the efficacy. Gene therapy but there are clinical trials ongoing. The vectors that are majorly used are lentiviral vectors and other associated vectors that were extremely successful on animal models even big animal models, and there are now clinical trials ongoing which are giving the first results.
Prof. Maurizio Scarpa: 00:37:32 Lentiviral vectors seem to be, together with hematopoietic stem cell transplantation, able to cross the blood- brain barrier and to differentiate and to produce enzyme. In the brain, adeno- associated viruses have been used mostly in the intra- ventricular, intra- parenchymal administration and they are expressing the enzyme. Adeno- associated viruses are extremely interesting because, I mean, they are vectors able to give a good expression of the enzyme and they are quite stable for a long, long period.
Prof. Maurizio Scarpa: 00:38:17 However, the major disadvantage of this is the production of antibodies and for this reason the need of having just one shot and potentially to have another therapy with another adeno- associated type if a second injection is forecasted. So, now what I said is a little bit the structure of what the neurological program can be seen in these patients. If you have any questions, please write and I can try to... So, there is one question. Is the papilla edema a reliable sign of endocranic hypertension? Well, in MPS, as I said, it is actually not a good sign.
Prof. Maurizio Scarpa: 00:39:27 The papilla is infiltrated with the gut storage and for this reason it is always more gray than the normal and it is a little bit more inflated or edematous. So, it is not really a very good sign to link to endocrinic hypertension. Is ERT modifying or treating the CNS progression? Actual ERT is made with enzyme administered IV. The enzyme is a high molecular weight, is about 40, 000 daltons. The blood- brain barrier is actually permissing a crossing to molecules that are less than 400 daltons. So, let's say at least 100 times less.
Prof. Maurizio Scarpa: 00:40:30 For this reason, the ERT is not crossing the blood- brain barrier at the moment and the only thing that we can do is to link the enzyme or to receptors, to markers that are recognized by a receptor like APOE or other molecules that are having a receptor on the blood- brain barrier or linking to nanoparticles or linking to, for example, there has been a company making monoclonal antibodies against insulin or transferrin. Is genotype, age, and effectiveness of therapy, individual factors, societal factors, affecting cognitive.
Prof. Maurizio Scarpa: 00:41:19 Well, this is a big question. I mean, genotype direct. Of course, if the gene contains longer deletions or stop codons can predict a severe phenotype. The age is sometimes important also to predict the phenotype. In the absence of other cases in the family, the sooner you diagnose the disease, the most probable the severity of the disease is big. Because I mean, of course, if you have a patient which can be recognized in the first couple of years in the first year, it means that it does have, he does or she does have a severity which is evident.
Prof. Maurizio Scarpa: 00:42:22 Individual factors or factors? Well, of course, I mean, being autosomal recessive with exclusion of the MPS2 which is X-linked, the fact that there is consanguinity can also be an important role. How do you define CNS disease? Can you predict which type will develop? Also, this is a very good question. Thank you. Well, to define the CNS disease is actually not so easy.
Prof. Maurizio Scarpa: 00:43:03 Of course, hyperactivity and sleep disturbances in very early age is, of course, predicting the severity of CNS. And for this reason, if you remember the slide that they were showing, the presence of hyperactivity, sleep disturbances, persistence of chewing and incapability of training, sphincteralial training are all signs that are actually prone to be in a severe patient. Is cognitive performance influenced by behavior, sleep disturbance and somatic manifestation? You're making difficult questions.
Prof. Maurizio Scarpa: 00:43:53 Of course, the cognitive performance can be indeed giving alteration to cognitive performance. What these children, of course, have is a disturbed sleeping hyperactivity. Sometimes they are having also acoustic problems, so they are deaf. And when they are sleeping, they never go to the REM phase. So the sleep is totally disturbed. And for this reason, they are under stress continuously. And this is, of course, giving also the cognitive performance alter. And so this is why we should take care very much about behavioral and sleep disturbance.
Prof. Maurizio Scarpa: 00:44:49 Can GAG accumulation give rise to inflammation like arthritis? GAG or any other substrate can indeed accumulate and give inflammation. But this is not a typical inflammation like in Rheumatoid Arthritis. The cytokines that are released are different. And indeed, one distinct sign in MPS at the joint is the presence of accumulation and alteration of the joint anatomy, but without the typical sign of an autoimmune disease. So it is giving inflammation but not autoimmunity in the joint. Okay, so I think that we don't have any other question. I thank you very much for the presence and for your participation.
Prof. Maurizio Scarpa: 00:45:57 I hope that this was useful. And I invite you to attend also the next webinar we will have next week. And I hope that this will be useful for your clinic and to be aware of these important diseases that now are having a lot of knowledge and possibilities of therapy. Thank you very much and have a good day.