Mucopolysaccharidoses (MPS) Case Studies

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Introduction

Professor Roberto Giugliani: Hello my name is Roberto Giugliani, I am a medical geneticist and Professor of Medical Genetics at the Federal University of Rio Grande du Sul in the South of Brazil. I work with lysosomal diseases in a reference center for lysososmal diseases which covers a large part of Brazil especially the diagnostic point of view and it is a pleasure to be here on the last webinar of this series in avoiding diagnostic delays in the diagnosis of MPS children and unravelling the mystery of MPS clue by clue.

So my the title of my talk is MPS case studies: practical examples of an effective identification referral process. So I will discuss with you some some Ideas I have about the how to improve diagnosis on MPS and have some clinical cases to to present and and discuss. So we will present around half an hour, and then we'll have time for questions I will show you; the questions should be posted in the chat, and then I will read the questions and they post their answers.

So please do not take any screenshots and do not reproduce any of the slides' content or images without express written permission of of the speaker. So as already, I will talk by about half an hour, and we'll have a Q&A session via text by the chat, and and play. And I will be very pleased to address your questions.

So let's move to the my disclosures. I have been involved in several, especially clinical trials on the MPS. Field and so the outline of my presentation.

Clinical suspicion of MPS

I'll talk about clinical suspicion of MPS, the importance of the early diagnosis, evaluation of urinary GAGs as a diagnostic tool, the enzyme assays we can perform, the molecular genetic analysis, and some diagnostic flowcharts, and I will end with 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 course.

There are exceptions to this; some small number of children may have hydrocephalus, and this may raise the possibility of MPS when you have this condition. But usually they are born normal, and the course is slowly progressive. Most patients present coarse faces 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 MPS III or MPS IV maybe mild or not apparent. Joint stiffness is also dominant in most types of MPS, although for instance in MPS IV, you may have joint laxity instead of joint stiffness.Or maybe none of the patients, or all of the patients.

So think about what you would say to this, to my question, would reply to my question, and I'll put the answer that in fact is none of these patients have MPS. So they are This the the one in the left. You see joint stiffness, but he has another condition called mucolipidoses. The patient in the center has a hepatosplenomegaly, but he has another condition called Niemann-Pick B disease.

And the patient in the right has a coarse face, but has another condition called alpha-mannosidosis. So the main conclusion is that you need the laboratory to make a diagnosis even with reasonable clinical suspicion. Let's go to a second question again: three faces. Which of these patients has MPS? So the 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.

Why early diagnosis matters

So let's move now to talking about importance of early diagnoses and my question is that easy to suspect MPS? For sure it would 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 say this is a normal child.

So depending on when the progress of the patient, you see this is how easy 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 siblings, one sister diagnosed at three years and a half, and her younger brother diagnosed at eight weeks of life. And you see the this so they they were evaluated at three years and a half.

The older sibling, the girl, when she was just diagnosed it, and the boy, uh, three years and a half after having enzyme replacement therapy. And you see the phases of the the two are completely different. You see the course phases on the left on the girl, and do not see this in the in the boy. But when you look at the spine, let's see the spine of the the girl in the left.

You see the scoliosis is very marked, and the boy is quite quite good shape. This is the spine, and you see the photos the same thing. They have the same genotype, the same enzyme deficiency but the difference is that the girl was not treated until 2. 5 years and the boy was treated almost since birth. So early treatment really makes a difference.

Take-home messages on urinary GAG testing

So my second take-home message would be the efforts to diagnose the MPS patient early in life. Let's see the evaluation of the urinary GAGs. And if MPS is suspected request urinary GAGs assay. This is very important and informative. Misleading results and avoid also qualitative screening tests like this, the toluidine blue spot test, or the citrate turbidity test.They can provide false positives.

That's not a major problem because we will make some tests to to clarify them. But can can give false negatives and this is a problem because you would miss this this patient. So avoid this. Do the quantitative measurement of urinary GAGs and use always use always use age-related reference values. I have another question to you: When increased GAG levels are found in urine, five options.

This result confirms that the patient has MPS. Second, MPS suspicion becomes stronger but confirmation is still needed. Third, the information is almost useless due to the high proportion of false negative false positives and false negatives. Four none above Five or of the above. So think ten seconds on these five options, and I will show the the answer.

So the answer is that MPS suspicion becomes stronger, but a confirmation is still needed. So the GAGs are screening; it's a screening test for mukopolysaccharidoses. The urinary GAGs you should move ahead, but it's not useless. It's important because all type almost all patients with MPS have increased urinary GAGs, but it's not confirmatory. You need to move ahead.

So second, be aware that some factors could interfere with measurements of urinary GAGs: some GAGs, proteinuria, and contamination. So it's very important that the urine is transported in 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 mucolipidoses, as multiple sulfatase deficiencies, as glycoproteinosis. So it's not specific for MPS. And there are also some false negatives that you may have with this test: GAGscretion decrease age. And also if very diluted urine is special. And also there is a variation of secretion of urinary GAGs along the day. So if you have a normal result in a patient that you suspect very strong suspicion, maybe you can repeat in another sample to to have a second result.

So my third take-home message is that 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 the 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.

Enzyme assays and the MPS types

One is the visceral-skeletal phenotype. They have CNS involvement and this occurs in MPS I, II, VI and 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 MPS IV, the Morquio syndrome, which may be subtype A or B.So, if you define one of these phenotypes, you can now be closer to a diagnosis.

One very important test that may help with the diagnostic process is the identification of the urinary GAG species because there are several GAGs present in the urine. The heparan sulfate, the dermatan sulfate, keratan sulfate so how they appear in the urine may give clues to the diagnosis. Isolated heparan sulfate, you say you see only 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 an MPS I, II, or VII.

So according to this pattern, you can focus your diagnosis and the next step for the diagnosis would be the the enzymatic. So and it's important too because as I showed before, you see all these faces; there are patients with MPS, but with different types of MPS. And it is important to identify the correct type because this is essential for to provide a a correct treatment and also for prognoses and genetic counseling and and prevention of future cases.

So there are at least eleven types of MPS, maybe twelve. 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 you have these types of MPS all the enzyme deficiencies you know. So but knowing the enzyme deficiencies you know exactly which type of MPS you have and this is because 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 then you progress in the degradation.

But any enzyme that is missing is deficient. You stop the degradation; you have storage of GAGs. So this is it's very important that all these enzymes work. In fact, the MPS investigation in dried blood spots is a practical sample for the assay of MPS enzymes. 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 easy to ship across countries, and this may be needed in some Sometimes, but assays in DBS are still taking as screening.

So patients with show positive results in DBS should be confirmed by additional biochemical or genetic analysis, maybe enzyme assays in in leukocytes or maybe a gene analysis in in the in the DBS to assay the enzyme activity. The most common is to use artificial substrate with this portion that is that may be that's not fluorescent when the substrate is is is not is integral.

But when you have a broke when you break the the bridge, you have you you with the enzyme you you have a fluorescence that is 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 you have the The fluorescent particles released, and you go to the fluorescent fluorimeter, and you you measure the fluorescence, and you see that 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 you are measuring. It's very it's really very convenient. 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 for the fifth talk take home message: All MPS patients have very low enzyme activity in respect of having the severe or attenuated forms. Another also, if low activity of a sulfatase, that some of the MPS enzymes are sulfatases. So we need to measure another sulfatase because there is a different condition called multiple sulfatase deficiencies. That is not an MPS, but you have deficiency of sulfatases and this, 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 a iduronate sulfatase deficiency. If you have a deficiency of iduronate sulfatase, please measure another sulfatase to see if it's not the multiple sulfatase deficiency. Is 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 patients, so there isn't any problem to differentiate this group. But in carriers, this might not be the case. You have overlap in the patients and controls so for carrier detection you usually need to go to the molecular genetic analysis to identify this patient.

From enzyme to molecular diagnosis

So sixth take- home message is enzyme assay is useful for patient diagnosis but it is not reliable for identifying carriers. For this you need the molecular genetic analysis. And we know all the MPS genes very well where they are, how many axis they have, what's the sequence, so we can sequence the genes that find the limitations. So in the case of MPS II we will sequence until you find a mutatio.

So in most cases and this is the 7th take home message, molecular genetics test should be performed to identify carriers. If the mutation in the case is known the lab should be informed to perform mutation targeted genetic investigation saving more time and cost. So if you have a family with a case and you already know the mutation you can say this to the lab and the lab will make a test for that specific mutation.

This is easier, quicker and less expensive. And it 's important to have the mutations because there is many conditions, there is a correlation between genotype and phenotypes. Or, for instance again in the case of MPS II we will have a neronopathic phenotype and patient is more severe. It usually pays when you have a major rearrangement in the gene versions, you have suspicion.

When you have misused mutations especially the ones that not interfered that much in the protein you have more attenuated. So it is important to know the mutation to predict this or try to predict. In addition to being important to the carriers, provide the genetical consulting to make easier prenatal diagnosis and so on. So the take home message is molecular genetic analysis may be useful for phenotype predictions and also for prenatal diagnosis and carrier detection.

Interactive questions

Let's go to the four questions. Which of these statements about MPS is correct? Urinary GAGs are increased in both patients and carriers. Two, very deficient in time activity is obserrving both patients and carriers. Three, genetic analysis allows for identification of carriers. Four, MPS diagnosis is only possible by genetic analysis. But the only way to identify carriers is by genetic analysis.

So the correct answer is three genetic analysis for identification of carriers. Why one is not correct because you know urinary GAGs are not increased in carriers, only in patients. Also the second one is not correct because deficiency in enzyme activity is not observed in patients not in carriers. And fifth is not common because 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 that linked the 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. So let's go to the flowchart. What's the usual diagnostic process? You 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 diagnose, confirm the mutations, and you have the the process completed. What is becoming more and more common now is you have a clinical suspicion, and then you go to the 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 assays or some for some of you. So you, but even if you have the molecular diagnosis or molecules, you need to go to the to do the enzyme assay to com to to confirm that you have these molecular variants your genetic variants you find found in the genotyping.

You they have impact on the protein so we need to do the enzyme assays and also that 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 you go to the molecular analysis to confirm the biochemical. So either way you do the testing is okay but you should do the complete picture at the end.

But there are several progresses on this and now we have some tests that there are very specific for each type of MPS. Different patterns and you can suspect the diagnosis with this kind of signature and this is becoming more frequent. So there are several ways to look for patients with MPS. One is departing from the clinical suspicion usually using biochemical screening.

We can also screen high risk groups like patients with short stature and then you use biomarkers like GAGs or gene panels to screen these patients and you have more patients detected. To detect all patients in the population, you need to do mass screening. The US has already recommended for MPS I and now for MPSII and I think this will become standard not only for US but for many countries in the world.

A case presentation

So now I will finish my webinar with a case presentation to illustrate some of the things I mentioned. So it's a patient from Brazil BOC, the patient born from normal non- consanguinous 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, showed mild thickening of mitral and aortic valves with no no systemic repercussion. After investigation by several specialists, MPS was suspected, and investigation was requested to our 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 A patient with suspected MPS, then they go to the doctor.

The doctor collects samples and send to the to the headquarters of the MPS Brazil network located in my city, in my hostel 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 direct to the doctor, and the doctor provides it to to the family.

So this and this we raise money from from several sponsors to maintain this program in order to provide the no these tests at no charge for to the family or to or to the doctor. And usually we start by measuring the urinary GAGs, and and in this patient we found an increased level of urinary GAGs. We then go to look at the species of ur of GAGs in the urine, and in this patient, we found heparan and dermatan sulfate, pointing to MPS I, MPS II, or MPS VII, and then you did the enzyme assays.

We discovered that the mother also carries the mutation can provide genetic counseling to the to this family and and also to other members of the of the family. And and you saw this patient was diagnosed very late, thirteen year old, and and this is a very common at least in Brazil, but it's common worldwide. And we made a study look at the the history for over one hundred MPS patients and what happens from the birth.

To the biochemical diagnosis, and you found that the symptoms they started around in average before 24 months of age. But the diagnosis takes almost five year, 4.8 years in average to to be reached. So it's a long delay, especially when we have specific therapies availables that provide a better prognosis to these patients. So it's very important to to to shorten this diagnostic journey of MPS.

And one one thing you know we observed on this study that most patients six seven on on 113 so they already had performed the 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, so to talk about about this. So the patient started ERT when he was diagnosed at 13 years old and this was good for him because he can improve the respiratory parameters, the sleep pattern, the walking, the liver volume, the urinary GAGs, the auditory tests as well. There are things that did not change with ERT so there are still unmet needs.

Echocardiography and visual acuity tests and also the enzyme replacement therapy did not improve the cognition because the enzyme does not cross the blood brain barrier. How will the diagnostic laboratory look like for MPS in the future? I think you will have a laboratory with just two equipments. A tandem mass spectrometer, where you can perform the GAGs and the enzyme assays and a sequencer, where you can find the mutations in all the MPS genes, and all of this you can do in dried blood spots.So this will be very much simpler than in the future.

Questions and answers

So summarizing. Let's see. If you go to clinical suspicion, to start of GAG, to GAGs and enzymes, 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 no, no not a shorter or quicker way to do this.

It mainly depends on what you have available. But I would say that the full picture is still is still important. Maybe in some cases that cannot have access to the to all the tests we may we may do a for instance if you have clinical picture you have increased GAGs then you have pathogenic mutations okay you can have the diagnosis if you have the Clinical suspicion, urinary GAGs, and enzyme deficiencies.

You also have a confirmation, but but then the way to they want to be quicker depending on your particular particularities of each one easier access to the molecular genetics or easier access to biochemical testing depends on the situation. But it is important to have the situation well confirmed to start therapy especially because therapy is very specific and you need to know which exactly type of MPS you have.

So there is a second question. Urinary GAGs screening tests, false positive or negative: how common are they? Very good question, depending on the test you use. You should avoid qualitative or semi-qualitative test and you should go to the measurement of urinary GAGs as a screening test. 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 on the second decade of life, especially sometimes MPS three or MPS IV. They may have some near normal values. But always take into consideration the the clinical picture and the interpretation of your results. There is another question: What do we have to do if you when you have a strong clinical suspicion of MPS?

If Positive GAGs and normal enzyme activity. So this is a very, very good questions that question we have so a suspicion, but you have a suspicion. GAGs are increased, but enzyme activity is normal. Well, I think that we should we should look for there are eleven maybe twelve enzymes involved. 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 diseases.

So you should do not give up on the investigation when you have a clinical picture in 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. 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 on MPS.

It's very important that we talk about MPS and you and we discuss the ways to make the diagnosis and enable these patients to have uh uh. Early diagnosis and the, and the appropriate therapy whenever possible. Thank you very much.

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