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[00:00:05] Dr. Christina Lampe: Welcome to the audience, welcome dear colleagues. We are today Professor Adriana Montano and myself will show you some complex case studies about mucopolysaccharidosis. It's a webinar four from a series of three by now. And these patients need a multidisciplinary approach. And we want to talk about what every pediatrician should look for to make early diagnosis. Just some housekeeping notes. Please do not take screenshots and don't reproduce any of the slides. We will have two case reports about 15 minutes and a question and answer session afterwards. And I would kindly ask you to ask questions at any point. We are the questions panel. So you can type your question and we will answer after our two case presentations. And please answer as much questions as possible. And I would like to start with my first case. My name is Dr. Christina Lampe. I'm working in Germany at the University Hospital of Wiesen, which is close by Frankfurt. And I'm taking care of lysosomal patients since 12 years. And actually, I don't want to present a young case, but a patient that is now 40 years of age. And she is a so- called attenuated MPS1, or Scheie patient. And as we all know, just to have a start up in this topic, MPS is a progressive disease affecting multiple organ systems. And not only the severe affected cases, but also the attenuated or more milder cases share the same signs and symptoms, but in difference of severity. And here, just to remind again, a spectrum of the disease severity in MPS1 from the very severe cognitive involved patients, so- called hurler patients. And on the other side, the patients without cognitive involvement, the Scheie patients that are seen more attenuated. And I would like to talk about the patient that you see here on the screen side. We have different therapies for these patients. We have the enzyme replacement therapy for all MPS1 patients. But in very young, severe affected MPS1 patients, up to 2 and 1 half years of age, we have also the opportunity for hematopoietic stem cell transplantation. And this is why it is extremely important to detect these patients and to transplant as soon as possible. I said already, we need a multidisciplinary team for these patients. And so a lot of specialties are involved around the metabolic specialist that is taking care of the patients. But now coming to the case. Kerstin was born as the first child of healthy, non-consanguineous German parents. She has actually one affected brother, but I will show you how late the diagnosis was made, and one healthy sister that is younger. She was born after an uneventful pregnancy, spontaneous, without any complications at 40 weeks of gestation. But she was quite small and of low height when she was born. And you see the pictures here. And maybe in this picture, you realize already that she is not very much flexible with her shoulder joints. And this is also what the mother realized in that child. In the first months of life, she suffered from umbilical hernia and received umbilical hernia repair. And at the age of three months, in a regular pediatric exam, she noticed some motor developmental delay, mild kyphosis, hypotone muscles, restrictions in abduction of both hips, contracture of the shoulders. So what do we have? We have already the stiffness of joints. And also the mother told the physician that she was suffering from recurrent infections of the airways, otitis, and also diarrhea. So the physician recommended a lot of physiotherapy according to the stiffness of the joints. But the mental development was normal. And you see her here from two, three months of age up to the age of three years. And she's looking like an absolutely healthy little girl. So what specialties she was visiting up to the one and a half years of age? And you see beside the pediatrician regular checkups, also the physiotherapist, the ENT, anesthesiologist, the orthopedics, and the surgeon. So between three and eight years, she suffered again from recurrent infections of the upper airway. And this is something very typical, unspecific, but typical for MPS. Recurrent infections of the airways, the ears, and also stiffness of joints. So this is very unspecific, but you have to think to MPS if you have a child like that. And actually she was much smaller than the others, and other children were teasing her because she was so small. At the age of seven, independent from the basic disease, MPS, because at that time nobody knew about MPS, she had a fracture of the left leg and she stayed in the hospital of two months. And this is why when she was a little bit stiff and walking and falling down quite often, everybody thought it is due to this broken leg. You see her here, the changes from three years to four years, five, and six years, and this patient. And actually you see she has not seen many more physicians at this time. Between eight years to 16 years, at 10 years, she had a stiffness and thickening of the fingers and also the the younger brother and the grandmother actually was very concerned about that because they were playing with cards and she was not able to move the cards in the hand very flexibly. She was very stiff. And so the grandmother recommended to go to a physician. So they went to a private practitioner and he suggested any skeletal problem and sent them to an orthopedics who made some x-rays of the hands. But she said everything is normal. So here on the picture on the left side, you see her with 14 years. And if you see the hands also with 10 years, you see that she has already quite stiff fingers or contractures of the fingers. With 13 years, the thickening and the contractures of the hands were more severe and she realized that she has a less mobility and the physician recommended physiotherapy in her. At the age of 16, the pediatrician or the private practitioner sent them to a specialized University Children's Hospital for skeletal problems and they made a CT scan, electrophysiology, and they also measured the enzyme activity in blood and they found indeed the diagnosis of an MPS-1 and the diagnostic was then confirmed by genetics. Actually, maybe you are wondering why there was no urinary gag done in this patient. It was, but the urinary gag was normal in her and also her brother. And this is quite typical for the attenuated cases that the urinary gag excretion in urine that should be increased in MPS patients can be normal in more attenuated cases or adolescents or young adults and this was the fact here. So, but fortunately they did the enzyme activity measurement immediately and they found the diagnosis. So from 8 to 16 years, you see that she also was seen by more specialties than before. Also, the orthopedic technician, the neuroradiologist that were part of the team taking care of her. Between the diagnosis and 31 years, she had no follow-up exams, no assessments, and no visits. Here, I have to say in 1996 when she was diagnosed, there was no treatment available. So for her and the parents, there was no reason to go to a specialized center or to check up for the disease regularly or the signs and symptoms of the disease regularly. So at this point, there was a desperate time for the patient because she was playing soccer quite often, but she realized that she was losing endurance and mobility over the years. And since the physician told her that at the age of 13 years, she will be wheelchair-bounded and paralyzed from neck to the toes, she was actually completely ignoring the disease and not thinking to that anymore because she was so scared to be completely depending on other people at the age of 30 years. So at this point, she was actually smaller than others. So she is 1.45 meters of height. She was always her whole life suffering from recurrent infections of the airways, pneumonia, and always long-lasting infections. And at the age of 25, she needed also glasses. So, if we see her from birth to 31 years, there was no treatment available at the age of diagnosis. The patient was not linked to a specialized center. She received some treatment that was done according to symptoms, but not preventive. Not all organs were checked, for example, not the spinal cord, for example, or the carpal tunnel syndrome was not checked. No contact to patient's organization. And in 2011, the patient by chance saw something about MPS and she connected to a specialized center and that's where I saw her the first time. So what do we have? We have the first symptoms at the age of three months. Then it took up to 16 years, up to the age of 16 years- to get a diagnosis, even if she had quite a lot of signs and symptoms of MPS1, like the stiffness of joints, the small body height, the recurrent infections, the hernia, but nobody was thinking to that and since there was no treatment available for sure, it was not something that was really forced to get. Then she had the symptomatic treatment, even if in 2001,, the enzyme replacement therapy was available. So five years after her diagnosis. But then in 2011,, she visited the specialized center and when I saw her the first time, we started making the check of all organ systems and this is extremely important in these patients to check the quality of life-reducing disease complications like the skeletal, the contractures, the eyes and the ears, but also the life-threatening disease complications like the CNS, the respiratory and the cardiac situation. And at the age of 31 years of age, when I saw her the first time, she had severe joint contractures, especially the fingers. She had also an aortic valve insufficiency- fourth degree and she needed surgery- and also a severe mitral insufficiency. She had craniocervical stenosis- at this time without indication for surgery- a mild corneal clouding but also an impaired visual acuity and recurrent severe infections with restrictive lung disease. Here you see now how many specialties she was seen by beside the metabolic specialist. So she started treatment at the age of 31 years of age and up to now we can say that she received a heart valve replacement in the same year when she was diagnosed. Her contractures of the joints are quite stable but she has severe pain in the right hip and you see the hip here with the hip dysplasia, especially on the right side. Her heart was stable up to now. Now she has a mitral insufficiency that needs surgery again. She had carpal tunnel surgery on both sides. The mild corneal clouding. She received in 2014 a cervical spinal cord compression. The lung has a stable pulmonary function and actually she's working as a teacher in a kindergarten full-time with babies and toddlers. So she's walking around with these children on her arms, she's living alone and does not need any support and she's doing quite a lot of physiotherapy to be active and fit and a lot of sports as she can do. So this is actually her. In summary, you see her in the first year of life. The diagnosis was 16, which is quite late in the start of ERT in 2011 and 2020, she is now in a very good condition. So conclusion one is the multisystemic disease need, indeed a multidisciplinary team. As you saw in this case. All organ systems need to be checked in order to prevent irreversible organ damage and not only by symptoms. They have a high anesthesia risk and need surgeries. So the multidisciplinary team need to discuss and balance the risk and benefit of surgeries and the best is to have a metabolic specialist who is the coordinator of that. So the planning and organizing of the necessary assessment is done by the metabolic specialist, discussed in a multidisciplinary team to understand what is needed for these patients, and then the results need to be discussed by the patient and for planning the next step. So this is actually very short. fast case, but I think I could show that the treatment of MPS1 is the best combination of or the enzyme replacement therapy available or the stem cell transplantation under the age of two and a half years in the severe affected MPS1 children. But not only that, also the regular follow-ups to detect the life-threatening and quality of life-reducing disease complications and an adequate symptomatic treatment for these patients. And whenever you see a child, a young child, with recurrent infections, hernia, and or joint stiffness, think to MPS and send the patient to a specialized center. And this all as early as possible. Thank you so much for listening. I will now hand over to Adriana Montano who will present the case number two. Welcome, Adriana.
[00:16:09] Prof. Adriana Montano: Thank you so much. Welcome, everyone. So as Christina was mentioning just now, one of the big challenges is to have an accurate diagnosis and early diagnosis for patients with MPS1. So today I wanted to share with you the experience of newborn screening for MPS1 in the state of Missouri where I live and the United States. So currently I am a professor from the Department of Pediatrics of St. Louis University, and I have also a secondary appointment in the Department of Biochemistry and Molecular Biology. So this is the outline of the presentation. I will talk a little bit about newborn screening in the United States, which is a little bit different than in Europe, the methodology that is currently being used, and then the specifics of the newborn screening of MPS1 in the state of Missouri and the United States, which will bring us to challenges and opportunities. Okay, so the childbirth statistics. So about 12 to 13 million births are happening every year in developed countries. So we expect to have from 400 to 500 patients with MPS per year, but usually we see much less. So the newborn screening in the United States is very complicated. So the March of Dimes and the American College of Medical Genetics would like to see that all babies in all states are screened for at least 29 disorders, and these 29 disorders are also called core disorders for which effective treatment is available. So in the U. S., each state is very independent, and they can have from 29 to even 80 different disorders screened. But at least the American College of Medical Genetics wants that at least 29 disorders are done. So disorders are divided into five different groups. So the amino acid metabolism disorders, organic acid metabolism disorders, fatty acid oxidation disorders, hemoglobinopathies, and others. So in 2009, Brady was born with Kravet disease. He was diagnosed at five months of age, and he passed away a few months later. So the parents of Brady decided to talk with the lawmakers from the state of Missouri and try to implement the Brady- Alan- Cunningham Newborn Screening Act. So the idea was that all babies would be screened for at least five different lysosomal storage diseases so they can be diagnosed early and treated early. So this act established that by July 1st of 2012, the Department of Health and Senior Services needed to expand the screening to include some lysosomal storage diseases. So initially, the proposed legislation would add the potentially fatal metabolic disorders of MPS1, MPS2, Pompe, Gaucher, and Fabry disease to the list of 67 disorders that were at that time being screened in the state of Missouri. There's some pilot studies. MPS2 was challenging to screen, so then MPS2 was dropped and Krabbe disease was added. So currently, MPS1, Krabbe, Pompei, Gaucher, and Fabry disease are being screened. So a little bit about the newborn screening in Missouri. So most of the babies are white with 15% of African American babies and some small numbers of Asian and American Indian babies. There are 3% of Hispanic babies, which can be white or black. So about 81, 000 babies are screened per year. On average, 400 specimens are tested per working day. And initially, 67 disorders were screened before 2012. After 2012, 5 lysosomal disorders were added and recently 2 more were added. So now they are screening for 73 disorders and they are proud to say that they save a baby every other working day. So let's talk about the methodology for newborn screening for MPSs. So historically, many scientists and researchers have been trying to develop new methods to start newborn screening for mucopolysaccharidosis. So initially, Dr. Hopwood started with the immune capture method where there was multiplex immune quantification assay for 11 different lysosomal proteins. So this method is simple, cheap, but the problem is that some patients may have normal protein levels but in inactive form. So this cannot be easily used for newborn screening. Then Dr. Gelb from Seattle developed a direct method assay for individual enzymes, which provides precise measurements but is very expensive. And we and others developed the GAG assay method where the GAGs are digested with enzymes into specific disaccharides and it's very good because you can run at the same time different samples and you can measure GAGs for all the NPSs at the same time. However, it takes a long time because we have to incubate the sample overnight and is expensive. So currently, what is being done for newborn screening in the state labs? So initially, the microtiter plate fluorometry, which has been used in Taiwan successfully, was developed by Dr. Chamolis from Argentina, where it is a simple enzyme activity assay in a 96- well plate, but it needs an 18- hour incubation time. This approach is simple, it's cheap, however, it needs a long time of incubation and it does not allow multiplexing. So currently, the state labs in Missouri in the U. S. use two different approaches. One is a digital microfluidic fluorometry where each enzyme reaction takes place in a discrete droplet on a disposable cartridge that incorporates reservoirs for five reagents. So five reagents mean that five different diseases can be measured at the same time and it can run 48 samples. So we will have four reservoirs for four MU calibrators, 40 reservoirs for 40 patient samples, and four reservoirs for quality control. This is done very quick, at low cost, and it allows multiplexing up to five diseases. Now the tandem mass spectrometry is also being used where there is an enzyme activity measured. It's good because you can multiplex, you can run different assays at the same time, however, it takes longer time and is expensive because you need a specialized technician and the maintenance of the machine is expensive. So, in Missouri, they are using the microfluidic approach. Here we see the state lab. They have 88 digital microfluidic platforms. The size is very small, it's the size of a PCR machine that you can put on any desk. This is a workflow, so they punch the samples. It takes about 15 minutes per 96- watt plate to punch all the dry blood spots. They extract the samples for about 30 minutes, and then they load the samples in the cartridge. The volume of the sample that is needed is about 3. 5 microliters. The running time is less than three hours. So how these microfluidics work is that there are some electrodes on a printed circuit board surface, and these are turned on and off to manipulate the droplets within an open or oil- filled chamber. So I have a small video to show you how the droplets are manipulated. So you see here the droplet that is being dispensed is transported throughout the cartridge. It can be merged, it can be mixed, and it can be split as well. So a little bit more about the cartridge, this is how it looks. So here we have the 40 samples, here we have the area of the reagent, so the reagents will mix with the samples, and we will have here the detection window. The reaction will be stopped, and here we will have the waste. So it's a very straightforward method. Now a comparison between the methodology of both digital microfluidics and the mass that is used in the U. S. So the digital microfluidics takes a very short time to process, so 30 minutes for extraction, loading time is five minutes, and the run is less than three hours. This was started in Missouri. Michigan already is using this methodology for newborn screening of MPS1, and Maryland is just starting to validate the technique. For tandem mass, we need an overnight incubation for 17 hours before it's added into the machine and is run, which is, the run is about three hours. So it takes a really longer time, but this was successfully implemented in the state of Illinois, and some other states already are using it. So this is the screening algorithm that is used in Missouri. So the samples are measured. If the levels and some activity of the hydronidase are low, are below the cutoff, and the cutoff is 1. 5 micromole per liter per hour, then they will retest the sample. If it's above that, then no further action is needed. Once they retest the sample, if both times the levels were below the cutoff, then immediately the state lab will contact the referral center, and the referral center will notify the primary care physician, the parents, and then they will coordinate, take another sample and rerun the test. Then the outcome will be communicated to the Missouri state lab. Here are some examples of the enzyme median activities that the state lab did. Here we see that the levels of the normal enzyme activity are dropped after the first week after birth, but they normalize after 14 days. But there was not much difference, and here there was no difference between gender. In red, you see the cutoff levels, so below this cutoff, then we expect to have a baby with possible MPS1, and here is borderline. So the state lab will keep an eye on the borderline levels of safety. So these are the results from four years of full population newborn dry blood spot screening in Missouri. So they have screened 308, 000 babies. They found 133 positive samples. Two of them were confirmed to have MPS1. So it's confirmed again by enzyme activity, and they do a mutational analysis. They found 71 pseudodeficiencies, which is true for other lysosomal storage diseases. They have found lots of people with low enzyme activity, but no clinical phenotype. And the incidence right now is 1 out of 154, 000 light birds. Currently in 2020, 22 states in the United States are screening for MPS1. So we see in blue the states that are screening currently for MPS1. And here we see a summary of the states that have screened the most. So we see Missouri and Illinois have the highest number of screens. And the incidence is about 0. 65 to 0. 45 babies out of 100, 000. The states of Washington, North Carolina, Kentucky, and New York are just starting the newborn screening process. So the incidence is misleading, so it's not accurate. So we need to have more data to really have an accurate number. And these bring us to challenges and opportunities. So having newborn screening implemented will give the chance to have early treatment. And at the same time, in the U. S., since the country is so fragmented, it can give us an opportunity to create a national registry and also to determine the precise incidence of not only MPS1, but other PSs. Thank you very much. And do you have any questions?
[00:32:58] Dr. Christina Lampe: Thank you so much. Adriana, I have the first question. In Germany, the newborn screening is divided by state. So the states decide which diseases they will put inside of the newborn screening. Is this from the government or the health care system, or is it in clinical trials that you are in studies that you have lysosomal diseases screened by newborn screening?
[00:33:22] Prof. Adriana Montano: It's the government who decides. So they have to make the case to the state person. They have just to make sure that there is treatment. And once the law is passed, then the state can start doing the screening.
[00:33:42] Dr. Christina Lampe: And it is without costs for patients, so it's covered by the health insurance?
[00:33:47] Prof. Adriana Montano: It's very minimum. I think it's about like $5.
[00:33:59] Dr. Christina Lampe: Here's another question. How many referral centers are in Missouri?
[00:34:05] Prof. Adriana Montano: So in Missouri currently, we have four different referral centers that the state lab can contact, and then they can reach the patients and communicate the results.
[00:34:24] Dr. Christina Lampe: So the next question is going to you, Adriana, as well. Seasonal temperature difference could affect the sample for newborn screening. Is that true?
[00:34:34] Prof. Adriana Montano: Yes. So it seems that high humidity and high temperature can lead to false positive levels. So that's why the dry blood spots need to be placed in bags with desiccant and placed and transported at minus 20.
[00:34:59] Dr. Christina Lampe: To minus 20?
[00:35:03] Prof. Adriana Montano: Yeah.
[00:35:05] Dr. Christina Lampe: Always? Or because we usually send the dry blood spot tests with room temperature?
[00:35:10] Prof. Adriana Montano: Well, room temperature would be okay, but at least a desiccant is important. And also they keep the dry blood spots at minus 80. So if they need to rerun, they can just do another punch of 3.5 millimeters and rerun.
[00:35:30] Dr. Christina Lampe: So there's another question, doesn't attenuated disease and MPS mean that patients might have less severe physical symptoms? I think it's a question to me and actually No, I think it is indeed that attenuated cases means that the progression is slowly, but it does not mean that they have less severe disease as you saw in the case she had cervical spine stenosis, she had the severe contractures, the reduced mobility and endurance. She has the pulmonary system affected and she has also the heart valve replacements of two heart valves. So attenuated means just slowly a progression, a slower progression, but not a milder disease. Any further questions? I think, Adriana, this is to you. Do you see differences in enzyme levels in premature babies?
[00:36:35] Prof. Adriana Montano: Yes, actually the enzyme levels in preemies is very different. They dry blood spots taken from babies that are less than 34 weeks provide unreliable results. So most of the time they have to re take the sample, reanalyze.
[00:37:00] Dr. Christina Lampe: But you do it then with dry blood spot as well or in full blood?
[00:37:05] Prof. Adriana Montano: In dry blood spot.
[00:37:09] Dr. Christina Lampe: Is hematopoietic stem cell transplantation also useful in older children or only up to the age of two and a half years? Actually, there are some countries where no enzyme replacement therapy is available. They perform also bone marrow or stem cell transplantations in older children. But what we know is that the transplantation is mainly working on the cognitive decline of these patients so we can stop the cognitive decline and the European recommendations say that up to the age of two and a half years there's the biggest benefit. If the child is older then you have to decide whether you use ERT or you will try the stem cell transplantation. But Adriana, how do you do it in the U.S.?
[00:37:56] Prof. Adriana Montano: Well, the same. Many, it's really up to the physician. So they recommend early stem cell transplantation because it's less risky. And also you can get better outcome. But you can see in some cases they treat with the enzyme replacement therapy early.
[00:38:23] Dr. Christina Lampe: So, how can I test for MPS? Gags in urine, DBS testing, enzyme activity in blood, genetics?
[00:38:31] Prof. Adriana Montano: Okay, well for MPS, the gold standard is first to look for gag levels in urine. So the typical way of doing this is to use dimethylmethylene blue. However, that gives lots of false negatives. So as you know, Christina was talking the gag levels were normal in her patient. So the most accurate way of measuring gags in urine right now is to use standard mass. And this is one, you know, most of the labs here are doing that. Then once you have high gag levels in urine, then you can measure in some activity for a specific you know suspected disease or suspected enzyme, which is the gold standard. After you have measured in some activity, then you can do mutational analysis to confirm.
[00:39:33] Dr. Christina Lampe: There are no further questions. Adriana, you want to say some last words before we close the session?
[00:39:44] Prof. Adriana Montano: Well, thank you so much. This is very exciting and if you have any questions, you know where to reach me. So thank you for being here today.
[00:39:56] Dr. Christina Lampe: Yes, thank you. Thank you, Adriana. It was a very nice and short while time now and very interesting. Thank you so much for listening. You know how to contact us and stay healthy.
[00:40:09] Prof. Adriana Montano: Bye-bye.