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[00:00:00] Dr Lucia Laugwitz: So, welcome to today's webinar on screening for metachromatic leukodystrophy. My name is Lucia Laugwitz. I'm coming from the Department of Neuropediatrics from the University of Tübingen in Germany. Thanks to the Initiative for Excellence in Pediatrics, we are invited today to share our clinical experience and our scientific work on screening for metachromatic leukodystrophy. So, before we start, please find here some housekeeping rules. Please do not take any screenshots. Please do not reproduce any of the slides, content, or images. You will have the possibility to submit your questions at any point, and we will have time to answer them after the presentation. Of course, you can find the recorded webinar available online afterwards. So, screening for metachromatic leukodystrophy. It is my pleasure today to introduce you briefly into the natural history of metachromatic leukodystrophy, focusing on the clinical course, especially on early signs and symptoms you might miss.
[00:01:21] Dr Lucia Laugwitz: I'll explain to you why the recent treatment options are now paving the road towards newborn screening approaches for MLD. I'll give you an update on the latest on newborn screening techniques, and in the end, we will talk about the chances and challenges that arise from the identification of pre-symptomatic individuals. So, today, we will be guided through this webinar by the cases of two siblings. We met the older boy at the age of seven and a half years when he was referred to our hospital. Going into his medical history, his pregnancy and birth were unremarkable. He achieved all developmental milestones in time. He had a normal development, and he joined kindergarten at the age of three years. Then, however, at the age of five years, he exhibited more aggressive compulsive behavior, erratic playing, and reduced attention span. So, he joined first grade in school, but the problems continued, and he was then diagnosed with ADHD and started on methylphenidate. A neuropsychological exam was conducted, and the IQ was 73, and he then revealed signs of developmental stagnation during the first year in school. And so, the neuropsychological testing was repeated a year later, and then he displayed cognitive decline with an IQ of only 52 then. So, at that point, the hospital decided to do an MRI, and we were consulted with the neuroimaging results. So, here you can see the MRI images. They clearly reveal T2 hyperintensities in the white matter. It's affecting the corpus callosum. You can see the typical triggered pattern for MLD, and they are clearly abnormal in comparison to age- and gender-matched controls. So, they're highly, highly suspicious for metachromatic leukodystrophy, and the little boy was referred to our hospital, and we completed the diagnostic workup. So, the family history revealed that he has a five-year-old sister that was reported to be healthy. The physical examination was unremarkable. The neurophysiology was unremarkable. The ultrasound revealed a slight thickening of the gallbladder, and the biochemical diagnostics showed a clearly reduced arsa enzyme activity in leukocytes and elevated sulfatides in urine, and then genetics confirmed the diagnosis with biallelic variants in the ARSA gene. So, of course, the most urgent question of the parents were, are there any treatment options available for this little boy? So, what we are talking about, it's metachromatic leukodystrophy. That is a lysosomal storage disorder. It's caused by a deficiency of the arylsulfatase A. Due to this deficiency, sulfatides accumulate throughout the body, but primarily in the central and peripheral nervous system, where they cause demyelination. Interestingly, the first child was reported in Tübingen about 100 years ago. So, to diagnose this disorder, we have the diagnostic triage, focusing on a deficiency of arylsulfatase A enzyme and accumulation we can detect in urine, and of course, the molecular genetics with biallelic variants in the ARSA gene. We do classify MLD based on the age of onset, and we do distinguish early-onset subtypes and late-onset subtypes. One can say the earlier the onset, the more rapid the disease progression and the decline of this fatal disorder. but no matter when the onset is, the clinical course is comparable in all subtypes. So the affected individuals show normal development and then they enter a phase of stagnation before they go into decline. They might exhibit variable symptoms. So the earlier the onset, the more likely the patients reveal motor signs as first symptoms. In the later subtypes, you have more behavioral and also cognitive signs during the first presentation. And there are some very early signs you might miss, as the acute onset, for example, of paralytic strabism, that might be first sign of late infantile MLD at a time point when the MRI is still almost normal. But when you look for gadolinium uptake, you can find it in the cranial nerves already. So this is a very early sign for late infantile MLD. On the other hand, early onset MLD patients might have normal MRIs when they're already symptomatic. So the CNS symptoms may precede MRI abnormalities in children with late infantile MLD. And they might be diagnosed with isolated peripheral neuropathy and misdiagnosed as long as the MRI is not suspicious for the classical pattern of MLD. On the other hand, in juvenile patients, there might be T2 hyperintensities long before they reveal clinical features of MLD. So no matter how many symptoms and early signs you remember of MLD, whenever a child displays a normal development and then enters a phase of developmental stagnation, this should prompt further diagnostics.
[00:07:19] Dr Lucia Laugwitz: And sometimes it's not MLD, but it's always an alarm sign to prompt rapid diagnostics. And you have to make a difference between a development that is always slow and a development that enters stagnation. So when you have a development that stagnates, you should prompt diagnostics and you should be fast. So when you then order, for example, next generation sequencing, and you already know this will take in your institute like six months, this is not fast enough. And you should think of further biochemical diagnostics and rapid genetic diagnostics. So why is this so important? We do have therapeutic options during the time of normal development. So this is our window of opportunity. And within the last 20 years, the allogenic hematopoietic stem cell transplantation has been established as an efficient therapy for metachromatic leukodystrophy for late onset subtypes. And just recently, we have good evidence on safety and efficiency of the lentiviral autologous hematopoietic stem cell gene therapy for MLD as well. So the stem cell transplantation is safe and efficient if patients are transplanted in an early symptomatic or even pre-symptomatic phase. It is not beneficial for patients with late infantile MLD. But for juvenile, especially late juvenile and adult patients, it is a suitable treatment option. And patients are really clearly improvement of survival, a clearly better gross motor function, and a clearly improved cognitive function. And you can remember, the earlier you treat, the better. If you're too late, the stem cell transplantation might trigger regression and a rapid deterioration of the patients. So, just recently, we got the approval for the gene therapy for early onset MLD forms. So, these are data coming from a monocentric study in Milan for early juvenile patients in the early symptomatic and pre- symptomatic phase, as well as for late infantile patients in the pre- symptomatic phase. And these data are very convincing. They show a clear improvement of motor impairment- free survival when treated early. And even more impressive is that these patients, especially when they're treated in the pre-symptomatic phase, show an almost normal cognitive development. So far, there are only a few treatment centers in Europe, and the gene therapy is now approved since 2020 for pre-symptomatic late infantile patients, for pre-symptomatic early juvenile patients, and for early symptomatic early juvenile patients, as long as they have a normal IQ and they show independent walking. And again, the data reveal the better the outcome, the earlier you treat.
[00:10:37] Dr Lucia Laugwitz: So, coming back to our case, we have completed the whole diagnostic workup. We see the clear hyperintensities in T2 in the MRI. So these are all signs of demyelination, and clinically, this child already shows sign of decline. So we cannot offer gene therapy. We can only offer supportive care. However, this child has a sister, and we did a full diagnostic workup on her as well. Her physical examination was unremarkable. Her IQ was 103. She had reduced ARSA enzyme activity in leukocytes and elevated sulfatides in urine, and molecular genetics revealed biallelic, likely pathogenic variants in the ARSA gene. And we continued to do an MRI, and interestingly, it revealed clearly hyperintensities in T2 of the white matter. So they're already signs of an ongoing neurodegeneration, although she's still clinically pre- symptomatic. But still, she has a good IQ. She's walking independently. So she is a candidate for gene therapy, and in fact, she was the first patient that we treated in Tübingen. And she's now one year out of gene therapy, and we're hopeful that her disease course will be much more favorable than her brother's. But of course, it would have been better to treat her before there are any signs of neurodegeneration on the MRI. And now having these treatment possibilities for early-onset and late-onset patients, we have two questions. Is this the best we can do?
[00:12:27] Dr Lucia Laugwitz: Identifying one sibling with symptoms, and then hopefully being in time for another sibling if the sibling is having the same genotype, to treat it. So a disorder with clear, efficient, and safe treatment options, and having a phase of normal development, this screams out for newborn screening approaches. So thanks to the group around Michael Gelb, there have been screening approaches biochemically improved and established that are available now for MLD. So we can do a screening from dry blood spots on sulfatides.
[00:13:15] Dr Lucia Laugwitz: We can do the ARSA enzyme activity from dried blood spots, and also genetic sequencing. So this is a two- tier approach with a third step of genetic analysis. So this sounds complicated. Why do we need three steps for this? Well, first of all, when you look into the data of this publication, you can see we have only 15 newborn samples. So, so far, we were not diagnosing MLD patients in the newborn phase. So we do not have a lot of biomaterial to define cut- offs and discuss thresholds. So here you can see there might be some newborns, especially with late-onset cases, who have quite low sulfatide levels. And as well, there are some normal individuals, so non-affected individuals, with quite high sulfatide levels. So to reduce the rate of false positives, we need the second tier, the ersoenzyme. And there are other special conditions you can find in MLD, for example, the ARSA pseudodeficiency. So there are polymorphisms in the ARSA gene that lead to a reduced enzyme activity and maybe a slight or no accumulation of sulfatides. So you might get these individuals by sulfatide screening and other screening. However, they remain healthy and they do not have MLD. They just have a pseudodeficiency. And the heterozygote individuals. So all parents, all carriers of these ARSA variants might exhibit low ARSA activity and sometimes a low accumulation of sulfatides. So to know whether you have, when you have slightly elevated sulfatides and reduced ARSA activity heterozygotes or pseudodeficient individuals, you need the genetic analysis of the ARSA gene. And furthermore, there are two disorders who mimic MLD biochemically. For example, the multiple sulfatase deficiency and the prosaposin deficiency. Both lead to an accumulation of sulfatides and multiple sulfatase deficiency.
[00:15:25] Dr Lucia Laugwitz: You also find a reduction of the ARSA enzyme and prosaposin deficiency in the ARSA might be normal, only slightly reduced. So unfortunately for these two disorders, there is no therapy available apart from trials. So we have to sequence the causative genes SUMF1 and PSAP as well, and take out of these positive rates, individuals suffering from other disorders than MLD. Despite these challenges, the screening introduced by Michael and his group is technically feasible. It has a low false positive rate when you go for the two tier strategy. The authors claim a near 100% sensitivity and a near 100% specificity. So this is a good screening approach. Other pilots are focusing on genetic screening of the other genes. Sometimes they include lots of other genes establishing next generation sequencing based newborn screening techniques. Some might argue this is the future in general, but for MLD, there's still a challenge. To date, there are more than 180 variants in the ARSA gene known to be pathogenic or likely pathogenic. But there are a lot of genes just not known yet. So any screening can only include pathogenic and likely pathogenic variants. So we will miss all the class three variants that are causative as well, and that we would cover when we go with a biochemical screening. And of course, as we cannot cover all variants, most likely because not all of them are established as disease causing and others are so new, they have never been reported in public databases. We cannot tell whether the sensitivity including only these limited numbers of variants is enough for a screening approach. However, we are very specific. So we are only screening for ARSA variants. We are not including other disorders. But to confirm these variants, we need the second tier of biochemical confirmation. So currently we are cooperating with a screening pilot project using a two tier approach, first screening for sulfatides and the second tier genetic screening. And so far we have identified three index cases. And we are conducting the confirmatory diagnostics as well as the treatment for MLD in Germany. So currently we are facing the challenge that the prediction of onset is difficult in newborns. So far we were only confronted with patients who are symptomatic and we need standards how to monitor these patients. So key to the prediction of onset is the genotype that has been reported in literature so far. We do know from our own data that the ARSA enzyme activity also clusters depending on the type of onset. However, you see clear overlap. Still you can say that very low residual ARSA enzyme activity leads to an early onset. And so we have the ARSA activity as a supportive parameter when predicting the disease onset.
[00:18:49] Dr Lucia Laugwitz: So, are there other parameters, especially in newborns, to predict the onset or to at least monitor them? Well, there have been some biomarkers reported that are useful for MLD, like the neurofilament light chain. So we know that it is elevated in symptomatic individuals, however, in pre- symptomatic individuals we just do not have enough data to tell whether it is early enough to be something that is useful for prediction. For sure, it is useful for monitoring, but also then you have to question whether something that indicates neurodegeneration is early enough to start the treatment, or you want to have parameters that are earlier than you can see or detect any neurodegeneration. Another parameter that's even less sensitive is N- acetyl aspartic acid, that increases neuron in symptomatic patients, while it decreases detected by MRS in the brain. So this again is a marker for neurodegeneration and we have data from symptomatic patients, but we do not have data from newborns and not enough data from pre-symptomatic patients to tell whether this will play a role for the prediction, but it can play a role for the monitoring. And of course, other techniques we're already using, MRI, MRS, neurophysiology, acoustic evoked potentials as well, they could all help us for monitoring, but they're probably not sensitive enough for the prediction of the onset.
[00:20:21] Dr Lucia Laugwitz: So in these three index cases, we were able to predict based on genotype and arousal activity the onset, we predicted in two cases an early onset, and both index cases are in preparation to already receive gene therapy, and in one case we would predict a late onset. So this patient is scheduled for hematobiotic stem cell transplantation at the age of five years. So why five years? Because then we are already early before the symptoms will appear for late juvenile patients, and we have a profile of minimal risks for the stem cell transplantation according to our hematologists. But of course, there are now all these pilot projects coming up, and we need common standards, we need harmonized processes, how to manage these patients and how to predict the disease onset. So there's currently a European consensus project started by Tubingen in Amsterdam, and I will give you some insights how we are developing such algorithms, how to manage these patients. So this is what we are currently doing in Tubingen, and the goal is to have standards that are the same, at least across Europe, hopefully also at an international level. So you have to define what you want to do with these patients, from the screening positive results, how you want to contact the families, what you recommend for confirmatory diagnostics to be really sure this is MLD, and at the moment we are discussing every case in international or European rounds during MLDI rounds, MLD cure rounds, to come to a common decision, what treatment we should offer to these families, and then we decide how to monitor these patients.
[00:22:12] Dr Lucia Laugwitz: So there's still work going on, and maybe a teaser for next year's webinars on a consensus, how to manage these patients. It is important to cooperate, to find these common standards, especially when we enter these new fields, and I think we as physicians are challenged to do so, to keep up with novel developments when we are implementing new therapies, new screening methods, and especially to keep up with the family's needs. So to sum it up, metachromatic leukodystrophy is a lysosomal storage disorder. After normal development, patients exhibit a developmental stagnation and progressive decline. Stem cell transplantation is a therapeutic option for pre- and early symptomatic late juvenile and adult patients, and we have the gene therapy that is a safe and efficient treatment options for pre-symptomatic late infantile and early juvenile patients, as well as for early symptomatic early juvenile patients. The prediction of disease onset still remains a challenge, and of course, newborn screening meets standards as well as the management of patients.
[00:23:31] Dr Lucia Laugwitz: Thank you very much for your attention, and we know we do have time for your questions. So we do have the question, what is the ratio of tested siblings that test positive for MLD? Well, this is based on the normal prevalence you have among the population. MLD occurs 1 to 40, 000 to 1 to 100, 000 at an estimated prevalence. Of course, it's an autosomal recessive disorder. So there is a 25% chance that another sibling is affected. So far, when we had no screening projects, of course, most of the patients we could identify for the studies of gene therapy, for example, going on in a pre- symptomatic stage, they were all siblings. So we have the interesting question when we will be able to publish our results of our research study. So I guess you mean the current consensus project that is going on, and we are currently submitting a Delphi to all experts among Europe. And we hope that we can submit our data in the early autumn this year to maybe publish it within this year and have at least among Europe common standards. So this is a very important question. When should we raise the topics of sibling screening and family screening with families on the first identification of symptoms or on the first child's diagnosis of MLD? Well, if we want to offer treatment to the siblings, if they are affected, we have to do the genetic and biochemical work up as soon as possible. So whenever one child is affected, we will talk with the family. Of course, it's an autism or recessive inheritance, and then we have to test other siblings because we can only, or the best window of opportunity to treat is when they're still in the pre-symptomatic phase. So as early as possible. And whenever there are already symptoms, we might be too late because the treatment, stem cell transplantation and gene therapy, it takes a year, a year and a half until you have the effect of the treatment. So we have to diagnose and to treat as early as possible. Are there NBS, DBS tests for MLD commonly available privately outside the EU and how do I source them? So far, there is right blood spot testing only available within pilot projects. There's no commercial way to buy these tests and to do them, it's technically challenging. It cannot be done in every lab. So also the newborn screening labs have needs certain equipment and need to change their equipment when they're now implementing MLD screening. So you cannot do it privately. It just can be done at the moment during pilot screening projects. For the five year old sister case, would she likely have followed the same trajectory as her brother? So with six to seven and a half year exhibiting signs of cognitive decline. So from our cohort studies and the German patient registry, we do have data that the disease course in siblings having the same genotype is comparable. However, there might be differences. And we even do see families where one child has an onset during the early juvenile phase and another one has the onset in the late juvenile phase.
[00:28:06] Dr Lucia Laugwitz: There are still factors we do not know about apart from the genotype. So normally you can compare the onset and you would expect a comparable course but it might vary in certain cases. So I do see no further questions. Thank you very much for your attention. I hope you can join the webinar tomorrow on treatment options in metachromatic leukodystrophy. It will be helped by my colleague Deepak Ram from Manchester. Thank you very much for joining. Goodbye.