SYNGAP1 in Translation: From Deep Phenotyping to Human Neurons

Here are our introductory comments:

Dr. Holder is an assistant Professor of Pediatric Neurology and Developmental Neuroscience at Baylor College of Medicine Houston.

He earned his MD-PhD at the University Of Texas Southwestern. His PhD focused on Genetics and Development. He then did his residency in pediatrics and neurology at Baylor College Of Medicine and post-doctoral fellowship in neurogenetics at University of California San Francisco. Dr. Holder is currently a neurologist at the Blue Bird Neurology Clinic and main investigator at the Holder’s lab at the Jan and Dan Duncan Pediatric Neurological Research Institute at Texas Children's Hospital.

The Holder’s lab's primary research is the genetic and neurobiological basis of neurodevelopmental and neuropsychiatric disorders. Their goal is to discover individualized therapies for neurodevelopmental disorders. SynGAP is one of the main areas of study. Using cell-based screens to mouse models of disease to neurons derived from induced pluripotent stem cells (iPSCs) of SYNGAP1 patients, they try to find genetic modifiers of the protein stability, to identify therapeutic entry points for SYNGAP1. His lab also has a focus on finding biomarkers and deep phenotyping investigating particular areas like sensory, sleep, and walking (waking?) patterns of SYNGAP1. Their ultimate goal is to translate the findings from the lab back to the clinic to improve the quality of life of children with SYNGAP1.

Dr. Holder has participated in several paramount publications in SynGAP and is considered one of the clinicians and researchers with deep knowledge of the disease. He is also extremely compassionate, enthusiastic, and kind when seeing his SYNGAP1 patients in his neurology clinic. We are truly grateful for the work he has done and his interest in SYNGAP1.

Transcript:

Thank you Mike and Marta for inviting me to give this talk today.

So a couple of disclosures: I will talk about some off-label uses of medications kind of a standard disclosure but I think the one that might be of more interest to the families that are on this call is that I'm actually a father of a child with a neurogenetic disorder which some of you may know and so that you know, in addition to my training, clinical training and basic science training, really informs me in terms of what it means to live day to day with a with a child with a neurogenetic disorder.

So for a number of years since I've been an independent investigator I've basically worked in these two buildings. The building on the left is the outpatient clinic at Texas Children's Hospital which is where the Blue Bird neurology clinic is and then the building on the right is the Jan and Dan Duncan neurological research institute and my initial goal was to take information to take knowledge, take samples, from patients that I see in the building on the left and take it to the right and try to understand at a deeper level the causes, the pathophysiology of neurodevelopmental disorders and then ultimately to, as Mike said, go back from the basic science realm, the translational science realm, back to the clinic with that knowledge and with the tools that we've developed to hopefully have individualized treatments for neurodevelopmental disorders.

So what I'm going to talk to you about today is some of our efforts specifically related to Syngap in that regard. So SYNGAP1 was first identified as a cause of neurodevelopmental disorders still just 11 years ago and it's really this landmark paper that probably most people on this call know about from 2009 where the first three children, three little girls with loss of function mutations were identified in SYNGAP1 with intellectual disability. Since that initial publication there have been a number of additional works that have identified mutations in other cohorts of children with neurodevelopmental disorders including epilepsy, intellectual disability again, as well as autism. Very recent work just from this year the largest study, exome study, exome sequencing study, to identify rare variants that cause autism identified Syngap as one of the most prevalent causes of loss of function mutations in autism. So what do we know about the SynGAP protein? So the SynGAP protein is a Ras/Rap GAP and that is it acts on small G proteins to regulate their activity. It normally acts to inhibit their activity and in particular for Ras inhibition of Ras results in downstream inhibition of signaling pathway that limits the insertion of a particular type of receptor called the AMPA receptor which is important for excitatory neurotransmission. When you have a mutation in Syngap and therefore a reduction in the SynGAP protein by 50%, you have an increase probably in the number of small G protein activity of small G proteins but this includes Ras which leads to increased activity of ERK and eventually increased insertion of AMPA receptors in the synapse. And overall this is thought to result in increased excitation of neurons and this leads to some of the phenotypes that we see in kids including epilepsy it's probably and probably also intellectual disability.

So with the knowledge that loss of function mutations in Syngap cause a neurodevelopmental disorder and 20 years of knowledge of the function of the SynGAP protein where do we go for patients with SYNGAP1 mutations? So what do we have? We have animal models including mice and rats and actually also zebrafish that model SYNGAP1 haploinsufficiency and based on that in 20 years of research into the basic neurobiology of SynGAP we know a lot about its function. Again we also have a growing list of human mutations, either loss of function mutations or other types of mutations called missense mutations which alter potentially either the stability of the SynGAP protein or the function of the protein.

We're learning more and more about the phenotypes that can potentially be associated with these mutations. And then very recently our group and I'll go through some of this data towards the end of my talk in collaboration with Gavin Rumbaugh characterize human neurons completely deficient for the SynGAP protein so what do we need although we know a lot about the phenotypes associated with SYNGAP1 mutations we do not currently have a large amount of prospective clinical data available that would be useful for future clinical trials mutations in SYNGAP1 are rare and it's likely that future clinical trials will utilize we'll need to utilize existing prospective data as as a control for determining whether a particular treatment is efficacious we need a better understanding of the heterogeneity of the clinical manifestations associated with SYNGAP1 mutations and this is in part through identifying more and more mutations in the SYNGAP1 gene but also in characterizing a larger group of individuals again in a prospective way with SYNGAP1 mutations we also need patient neuron data this data will be extremely useful for validating whether treatments that are developed in the lab are potentially efficacious in human neurons we need therapeutic entry points and we need preclinical studies of those therapies developed in the lab so what I'll talk about today are some of the some of the work that we've done both in the clinic as well as in the lab looking at the heterogeneity of clinical phenotypes associated with SYNGAP1 mutations some of the work that many of you on this call are listening to the recording have been involved with in terms of prospective clinical studies of phenotypes associated with SYNGAP1 mutations.

I'll talk some about the our efforts in the lab to develop patient-derived neurons for investigating pathophysiology as well as for therapeutic modeling and then at the end I'm going to talk about some potential approaches for personalized therapies for patients with SYNGAP1 mutations including things like gene therapy so this is a an update of a figure we published a couple of years ago now demonstrating the landscape of mutations found in SYNGAP1 associated with neurodevelopmental disorders and I will say that the number of publications the number of patients identified with mutations in Syngap is growing so fast that it's hard to keep up with this figure but one thing that you'll notice is that the majority of patients with mutations in SYNGAP1 those mutations tend to land towards the middle of the gene basically from exons four through 18 or so of the gene and the reason for this is probably because there's extensive what's called alternative splicing that occurs at the far ends of the gene and what that means is that although SYNGAP1 is a single gene that single gene can produce a number of different types of protein of proteins and mutations that occur more at the five prime at the very far left or the very far right of the SYNGAP1 gene potentially only affect a small subset of the proteins that are made from this from this gene and therefore may not have as as significant deleterious effects as those mutations that land right in the middle of the gene.

So again about five years ago I began seeing patients with SYNGAP1 mutations and began characterizing their neurodevelopmental trajectory and their neurologic phenotypes and one of the first things that we did was was very simple which is just what we do routinely in clinic just to ask about development and ask when a child with the SYNGAP1 mutation sat unaided when they walked unaided when they said their first word etc and began compiling this data and what we found was that for all of the developmental milestones that we asked about there are significant delays on average in kids with SYNGAP1 mutations so you can see in this chart that the average age of first word said is around 36 months or so for our cohort of SYNGAP1 mutation patients whereas the neurotypical kids say their first word somewhere between 10 and 12 months our kids with SYNGAP1 mutations sit unaided around 9 months 9 to 12 months or so on average this is delayed from neurotypical kids which is around 6 months our kids walk a little bit later the neurotypical kids walking in about 22 to 23 months on average and then fine motor skill development is also significantly delayed in terms of being able to scribble with a pen or pencil and using utensils so this is this is basically retrospective data asking parents to recall when they perform these milestones for the first time and we really wanted to do this in a more rigorous way and so to do this we began I began collaborating with developmental pediatricians at Texas Children's Hospital to longitudinally record development in children with SYNGAP1 mutations and the tool that we use is something called the caputi scales this is basically a play assessment where we have these toys shown here on the left we ask the the children to perform certain tasks things like this form board here where they just place a circle a triangle and square in the in the board stacking blocks drawing lines etc and we do this every year for our children with SYNGAP1 mutations to in a longitudinal way see how children with SYNGAP1 develop over time.

And so we've been collecting this data again for the past five years this is an update from our publication in 2019 and what we found was really for all domains of development that on average our SYNGAP1 kids have are significantly delayed compared to neurotypical kids so just to orient you to this graph here on the left the y-axis here are h equivalents that's basically how a child would SYNGAP1 scores on gross motor development and the x-axis here is the chronological age of the child so for a neurotypical child let's say at 50 months on average neurotypical children should score an age equivalence of about 50. And what you can see for our SYNGAP1 kids for gross motor development there's a significant delay again at 50 months and really at every age compared to neurotypical but what you can also see is that children with SYNGAP1 mutations continue to learn throughout their lifetime. So on average the age equivalents for our SYNGAP1 kids increase in terms of gross motor development over time and that's why it's vitally important that while we're waiting for the development of these targeted therapies for children with SYNGAP1 mutations that all children with neurodevelopment disorders including SYNGAP1 continue to receive the therapies that can best enhance their development and as I'll show you in terms of gross motor, fine motor as well as language development. Shown here on the right is what's called the developmental quotient. This is basically taking the age equivalence dividing by the chronological age and what you can see is that over time this line on the right goes down. The average developmental quotient. This does not mean that children with SYNGAP1 mutations all regress uniformly at all. What this means is that the distance between where a child with the SYNGAP1 mutation scores and that of neurotypical kids widens with time.

So not only are children with SYNGAP1 mutations delayed but there is a reduction in the tempo by which learning occurs over time. However it's very important to realize that learning continues throughout the life, throughout the childhood of these kids as we see here on the left with improving age equivalence on gross motor development. Same is true for fine motor development. I won't go into so much detail but again we see a delay in fine motor development on average in our kids with SYNGAP1 mutations and then also for language development as well. One of the things that we've noticed and you can see this really most clearly here for language is that while on average there's a significant delay in language development for kids with SYNGAP1 mutations there's really quite a large scatter and the reason for this large scatter is not really clear. We're looking into whether the location of the mutation in SYNGAP1 may play a role and whether a child is higher on this curve than other children with similar mutations in other parts of the SYNGAP1 gene, whether other factors play a role and this is work that's ongoing in my group. So delayed development is certainly one of the hallmarks of mutations in SYNGAP1.

The second very common phenotype are seizures. At least 90% of children with loss of function mutations in SYNGAP1 have seizures greater than 90% in the cohort that II follow at Texas Children's Hospital have seizures and we looked in detail as to what type of seizures these kids have. And so in our cohort and others that have been published as well, we see that atypical absence seizures tend to be the most common type of seizure identified. Greater than 60% of the kids that I see in clinic report having atypical absence seizures. So these are seizures they're basically associated with staring spells and they can also be associated with things like eye fluttering or eyelid what's called eyelid myoclonia. In addition to atypical absence seizures we also very frequently see atonic seizures in our kids with SYNGAP1 mutations. These are drop attacks. These are very short but can be very devastating seizures because the children have complete loss of tone and that can result in significant injuries depending on where the child is when the seizure occurs. We also fairly frequently see myoclonic seizures and sometimes generalized tonic clonic seizures and again sometimes these seizures occur in combination, so an individual child with with a SYNGAP1 mutation will not necessarily have just one type of seizure but they but may have multiple types of seizures. Most common are atypical absence seizures and atonic seizures. In fact these can occur in very close approximation. So shown here on the right is a video from Ingrid Sheffer's group in their paper in Neurology and what you can see in this video is that this child has an atypical absence seizure with eye fluttering and that suddenly converts into an atonic seizure where she has loss of tone and you also notice that this is associated with eating which seems to be a fairly common occurrence in children with SYNGAP1 mutations where they can have what's termed a "reflex seizure". That is a seizure in response to chewing or eating.

So since seizures are so common what medications work really well for children with SYNGAP1 mutations? And to really understand this in the best way we need prospective data looking at individual medications as to whether they are efficacious in a blinded fashion in patients with SYNGAP1. But what we did is just looked retrospectively at what medications children that I see in clinic are taking and whether the parents report that there are efficacious or not and for whether a medication is effective we use a commonly used standard which is the seizure burden reduced by at least 50 percent. So what you see in my clinic is that the most commonly prescribed medication that I found to be efficacious is lamotrigine. Many of my patients take lamotrigine and see a positive effect. Although you will see that while two-thirds of patients on lamotrigine have some reduction, have significant reduction in their seizure burden, there are about a third or so of kids that either do not have a significant reduction in their seizure burden or have significant side effects associated with lamotrigine. In addition, the benzodiazepines which include things like clobazam and clonazepam seem to be particularly efficacious in our group of patients and then there are some medications that appear to particularly not work well in kids with SYNGAP1 and these include things like the sodium channel blockers, oxcarbazepine as well as lacosamide and rufinamide.

So again this is retrospective data looking at the medication use in kids that I see. Going forward what we really need is more prospective data looking at these medications both in terms of their effectiveness in seizures as well as in other phenotypes in kids with SYNGAP1 mutations. So since patients with SYNGAP1 mutations so frequently have seizures they also frequently undergo EEGs and these are, you know, usually outpatient EEGs that last for about an hour. Occasionally kids that I see will have longer EEGs to determine the total seizure burden and to characterize events and determine whether they are in fact seizures or not. I just want to show you an example of an EEG here on the right. This is an EEG from a neurotypical individual. See all of these squiggly lines from different leads that are placed on the scalp shown here on the left and some of the abnormalities that we see in kids with SYNGAP1 mutations most commonly, the most common abnormalities seem to occur in the back of the brain or the occipital region. We don't really know why this is and this seems to be a very unique feature of the EEG abnormalities that we see in kids with SYNGAP1 mutations. Shown here is an EEG in a child with SYNGAP1 mutation who is having an atypical absence seizure. So in the first part of the tracing here you see really what is a normal EEG pattern with low amplitude high frequency waves and then about 10 seconds or so into this recording you see suddenly the EEG changes dramatically where in basically every lead you see these high amplitude low frequency waves that were associated with the behavioral arrest and this behavioral arrest is an atypical absence seizure with freezing of behavior and eyelid myoclonia. Another feature that's not associated with seizures but that we do see fairly frequently in kids with SYNGAP1 mutations are these occipital intermittent rhythmic delta activities. So shown here this tracing looks somewhat similar in that you see these high amplitude low frequency waves but in this case you really only see them in a few of the leads, not in every lead and what's interesting here is that the child from which this EEG was recorded was behaving as they normally do. So despite having this markedly abnormal EEG, this did not result in a seizure and so again we don't understand why this particular pattern seems to be prevalent in kids with SYNGAP1 mutations. SynGAP is expressed all throughout the brain. It's not just expressed in the back of the brain. So why exactly we see this pattern we don't really know but this is a very typical pattern for kids with SYNGAP1 mutations and almost pathomonic for these kids.

So we've talked about development. We've talked about seizures. Behavioral abnormalities are also very common in kids with SYNGAP1 mutations. In our group of patients we see most commonly aggressive behaviors in our children followed by self-injurious behaviors as well as hyperactivity. I also included in these behavior abnormalities elevated pain tolerance which is very high. About 50 percent of parents report that their children have elevated pain tolerance where things that should really cause significant distress really do not cause issues and then about 50% of the kids in our cohort take medications because of the behavioral abnormalities and some of the medicines that we use in my clinic, and again this is retrospective data, include things like guanfacine and clonidine. These are both originally developed as blood pressure medicines. They don't actually work that well for blood pressure but have been found to be useful for behavioral outbursts, for hyperactivity behaviors and they seem to work fairly well. Stimulants have been tried in several of the kids that I see with SYNGAP1 mutations. Typically seem not to work. In fact they... most of the kids that I see with SYNGAP1 mutations have the opposite effect that you would expect from a stimulant where they become much more hyper or much more aggressive and then a few of the kids that I see also take risperidone which is a antipsychotic medication also approved for behavioral issues in kids with autism. So I'm going to focus on this elevated pain tolerance a bit more and more specifically about sensory processing in general.

So in 2018 Gavin Rumbaugh's group published this paper where he looked at sensory processing in mice that are heterozygous for a loss of function mutation in Syngap and it's a beautiful paper, very in-depth investigation of this but I just pulled out this one piece of data to show you which where basically what he found was that mice with a mutation in the SYNGAP1 gene are not able to effectively discriminate between two different tactile substances. Two different substances. So shown here in Panel E basically what you see is that in wild type mice they're able to very effectively distinguish an object that they've seen before versus a novel object. In contrast the SYNGAP1 mutant mice are unable to distinguish the two different objects, the object that they've seen before and the novel object. We participated in this not really in the mousework but in identifying a number of individuals with SYNGAP1 mutations whose parents reported that they had significant sensory processing issues and this included things like avoiding loud sounds or seeking certain sensory inputs but this again was really in a retrospective way, just looking through charts, asking parents to give their impression of their child's... overall impression of their child's response to novel stimuli. We wanted to do this in a bit more of a rigorous way and to do this we use something called the Sensory Profile 2. This is basically a structured questionnaire where we ask about different sensory inputs, things including whether children seek certain sensory inputs, whether they try to avoid certain sensory inputs, whether they have a particular sensitivity or lack of sensitivity to certain inputs etc. and we did this both in two groups of patients that I see that includes individuals, children with SYNGAP1 mutations as well as those with a related disorder called Phelan-Mcdermid syndrome and we did this just to see whether these two disorders which in many ways are very similar in terms of development in terms of both increased risk for seizure and then also the proteins that are responsible for these two disorders really work kind of hand in hand in neuronal communication. So we were interested in whether for sensory processing whether there were similarities or differences between these two neurodevelopmental disorders. And what we found for both Phelan-Mcdermid syndrome and for our kids with SYNGAP1 mutations is that there is remarkable... there were remarkable abnormalities in sensory processing in both of these groups. So for each of these graphs the black bars represent individuals with Phelan-Mcdermid syndrome, the blue bars individuals with SYNGAP1 mutations and then the green shaded region here represents the scores of typically developing responders. And what you can see is for each of these domains that we measured seeking sensitivity avoiding and registration of novel sensory inputs that on average kids with both Phelan-Mcdermid syndrome and SYNGAP1 mutations have significant abnormalities in their processing of sensation.

So similarly to sensory processing, a large percentage of our kids that I see in clinic and that have been reported in the literature with SYNGAP1 mutations have sleep abnormalities. This seems to be greater than 50% at greater than 50 percent of kids with with SYNGAP1 mutations. So we want to again look at this in a rigorous way and to do this we used a another tool called the Childhood Sleep Habits Questionnaire where we again compared kids with mutations in SYNGAP1 versus Phelan-Mcdermid syndrome versus healthy controls. And what we found was that our patients with SYNGAP1 mutations their overall score was significantly higher on this structured questionnaire compared to healthy controls and actually compared to those kids with Phelan-Mcdermid syndrome who also, whose parents also frequently report abnormalities with sleep. So just to orient you, for total score a score of 41 or less is considered to be a child without significant sleep abnormalities and a score greater than 41 the child has significant sleep abnormalities. So again you see that kids with SYNGAP1 mutations on average have a significantly higher score than the healthy controls and significantly higher than this cut-off score of 41. So what's driving this overall score? The sleep questionnaire measures multiple potential abnormalities in sleep including things like night awakenings, sleep duration, sleep onset etc. and what we found for our patients with SYNGAP1 mutations is that nighttime awakenings, sleep durations, and things called parasomnias and these are things like restless leg syndrome, all of these seem to be significantly worse. The scores for these categories seem to be significantly worse in kids with SYNGAP1 mutations compared to healthy controls.

We wanted to look at this in a bit more detail to see whether the age of the child for which the parents were answering the questionnaire impacted overall score of any of these domains and what we found was that overall for SYNGAP1 kids there really was not much difference whether the child was under 10 years of age or over 10 years of age and this was actually different than what we saw for Phelan-Mcdermid syndrome where younger children seem to have more typical sleep patterns and older children seem to with Phelan-Mcdermid syndrome develop those worsening sleep patterns and this was and when you look at the score of individual domains what we found is that for some domains such as for parasomnias we again saw that whether a child with a SYNGAP1 mutation was under 10 or 10 years or over a similar pattern in the reported frequency of parasomnias. In contrast there were some areas where the sleep pattern seemed to be different in SYNGAP1 kids whether they were under 10 years of age or over 10 years of age. So for example in sleep onset delay those kids under 10 years of age didn't have more sleep on set delay than those kids over 10 years of age with SYNGAP1 mutations. In contrast for sleep anxiety those kids under 10 years of age tend to not have... parents did not report significant amount of sleep anxiety on average compared to those kids over 10 years of age where there was a significant amount of sleep anxiety.

So the next thing that we're doing in terms of sleep is to look now in a more prospective way using something called actigraphy at sleep patterns in kids with SYNGAP1 mutations. So actigraphy basically uses... it's kind of like a smart watch that measures movement in children both during the daytime and while they're sleeping.
Movement of course is reduced significantly during sleep and the watch can, the software can record when sleep's occurring which is shown in these light blue areas of the recording compared to wakefulness and active wakefulness which is shown with this yellow bar showing activity. And what we found overall for kids so far (and this study is still ongoing) what we've found so far is that our SYNGAP1 patients while they do on average actually have fairly normal sleep content that their sleep is inconsistent. So what you can see in the first day here is that this child with a SYNGAP1 mutation only slept about four or five six hours or so. The next day they slept about eight or nine hours and then down at the bottom there was a night where this child only slept three to two to three hours or three to four hours or so. In contrast their neurotypical sibling you can see that every night their onset of sleep, their offset of sleep, their sleep duration is fairly consistent. So just to show you that in terms of numbers if you'll focus here in this middle column, the total sleep, if you look at the average, the average sleep for the SYNGAP1 patient was actually pretty normal. It's about eight hours and actually quite similar to their neurotypical sibling but then if you look in more detail at the consistency of their sleep the minimum amount of time they slept was only about three hours and then the maximum time that they slept on a given night was 12 hours. In contrast the neurotypical sibling is much more consistent sleeping between six to eight hours every night and so, you know, this just shows that while on average using this sort of tool this child may sleep in normal... have a normal sleep content, on any given night you really don't know whether this child is going to have a good night or a bad night whether they're going to sleep a short amount of time or have a good sleep pattern.

Okay. So I'm going to move on to the next study that were that we've been working on now for a number of years which is to really look in some detail at gait in children with SYNGAP1 mutations. So as I mentioned very early in my talk, gross motor development is significantly abnormal and significantly delayed in kids with SYNGAP1 mutations. But in addition to that even once children with SYNGAP1 develop are able to walk or able to run, their gait as reported by parents or is observed by neurologists is significantly different than that seen in neurotypical children. But we wanted to look at this in a more quantitative way as a potential clinical endpoint and to do this we're using something called motion capture gait analysis and what this is is basically having these reflective balls placed at various joints on an individual either with a SYNGAP1 mutation or a neurotypical child and they're recording gait or walking either on over a carpet, over a flat surface, or on a treadmill and then various
data points are recorded using an array of cameras placed around either the carpet or the the treadmill. So this is what the gait lab looks like. This is a flat carpet here that we have children walk over usually around 20 times or so. This carpet is actually not just a simple carpet. It has sensors in it that allow us to detect things like the force with which a heel strike occurs, the length of a stride etc and then those reflective balls which are recorded by the array of cameras also allow us to look at things like joint angles and then so we do the gait analysis again both on this carpet as well as on a treadmill. This treadmill is also not a just a typical treadmill. It can also record things like force of heel striking and things like that. And so this is the sort of raw data that we get from these studies. Basically shown here in this top panel you can see the individual reflective balls are now these red and gray balls and that are connected by lines from the hip down to the leg, the knee and the foot and basically using this system we can record again things like stride length, time it takes for individual strides, joint angles etc. and then the overall goal here is to compare a cohort of kids with SYNGAP1 mutations with neurotypical controls.

So shown here is just data from a pair of children from a single family, one child with a SYNGAP1 mutation and their neurotypical sibling and what you can see is you can record things like mean stride times again things like mean stride lengths. We're just beginning to look at this data unfortunately, I know a lot of you are interested in the study and some of you have participated in this study, the gait lab has been shut down because of COVID but we're hopeful that in the new year as vaccines become available and our infection rate goes down that we'll be able to reopen the lab and collect the rest of this data. So what does this all mean in terms of in terms of patient outcomes? So we wanted to look at this using something called health health-related quality of life and this again is a structured way of looking at various domains that contribute to quality of life and these include things like school performance, physical performance, emotional well-being, psychosocial performance and socialization and so we looked at again at our patients not just the patients that I see in clinic but actually patients from around the country with SYNGAP1 mutations and compare them to individuals with other neurodevelopmental disorders including Rett syndrome and Phelan-Mcdermid syndrome again as well as published data from individuals with idiopathic autism, idiopathic intellectual disability as well as other chronic health conditions and healthy controls.

And what we found using this study, using this tool is that kids with SYNGAP1 mutations parents report significant impairment in their health-related quality of life compared to healthy controls and actually similar to those individuals with other syndromic neurodevelopmental disorders such as Phelan-Mcdermid syndrome and Rett syndrome and the scores seen in the kids with SYNGAP1 mutations and these other syndromic autisms is actually significantly lower than those of idiopathic autism as well as idiopathic intellectual disability. So we wanted to dive into this a bit more and we looked at the individual domains that are measured by this health-related quality of life tool and what we found is that for some of the domains such as for socialization our kids with SYNGAP1 mutations with Rett syndrome with Phelan-Mcdermid syndrome have similar impairment as those individuals with idiopathic autism with idiopathic intellectual disability. What is really driving down the score of these for these kids with syndromic autism is their physical performance. So individuals especially with Rett syndrome but also with SYNGAP1 mutations as well as Phelan-Mcdermid syndrome have significantly more impairment in their physical well-being than those kids with idiopathic autism or idiopathic intellectual disability. I think that just goes back to the earlier data that I showed you where these kids, especially with SYNGAP1 mutations, have a significant delay in development of their gross motor milestones and then again even after those milestones are achieved continue to have impairment in their gross motor functioning and their fine motor functioning. Okay.

So I'm going to change gears now and move from the clinical realm more to the basic science realm. So we've been interested for a number of years in developing human neurons from patients with SYNGAP1 mutations as well as from other neurodevelopmental disorders to try to understand better the pathophysiology that leads to the phenotypes we see in patients and also as a platform for potentially validating interventions that we identify in the lab that eventually we would like to translate to the clinic. So to do this for SYNGAP1 patients we've done this now for a number of patients with SYNGAP1, we simply draw blood from our patient, isolate a particular cell type in the blood called the PBMC. We then take those PBMCs expand them and infect them with a particular type of virus that leads to expression of what are called the Yamanaka factors. These are factors that allow for somatic cells, that that is cells that have terminally differentiated or now blood cells and are always going to be blood cells we were able to reverse their differentiation and get them to a more immature state which is a stem cell state. And then from those stem cells we're able to differentiate those into anything we want in the lab and of course for SYNGAP1 we're interested in developing neurons and investigating the impact of SYNGAP1 mutations on neuronal function. So again this just shows us sort of in picture form what we do. We expand these PBMCs. We reprogram them to develop stem cells. We do quality control with these stem cells to validate that these are in fact what we call pluripotent that is they can develop into any type of cell that we want and then we differentiate these into neurons. Importantly for for these type of studies we have to not only create stem cells from, derive them from patient blood but we also have to create the proper controls for any sort of experiments that we do.

So for these type of studies we have to correct the mutation that is present in the stem cells that comes from the patient in SYNGAP1. Snd so to do this we use something called CRISPR gene editing. This basically allows us to hone in on the mutation that is present in the patients in the stem cells that we've differentiated from blood in SYNGAP1 and convert it back to the wild type sequence. So this is actually... although I kind of show it here in one slide maybe it looks fairly simple this is actually by far the most arduous part of these studies is getting these corrected stem cells so that we can differentiate neurons from the patient and then from the corrected stem cells to directly compare. And so we've done this now for one of the patients that we collected blood from and generated iPSCs and some of that data shown here just showing that we were able to correct the mutation.

We're now, these stem cells are undergoing additional quality control experiments including things like exome sequencing to make sure we didn't introduce mutations elsewhere in the genome as well as some of the other quality control work that we do with these stem cells but while we were generating these stem cells from patients with SYNGAP1 mutations we began a collaboration again with Gavin Rumbaugh to look at neurons that are, human neurons that are completely deficient for SynGAP. That is that they have mutations in both copies of the SYNGAP1 gene. So his lab, again using CRISPR gene editing was able to introduce mutations into one of the exons of SYNGAP1 into exon 7 where many human mutations have been identified. He introduced mutations again into both copies of the SYNGAP1 gene that resulted in complete loss or nearly complete loss of the SynGAP protein and that resulted shown here on the right again by protein by western blotting in greater than 90 percent reduction in SynGAP protein. So he sent us these cells to begin to look at neurophysiology, to try to understand whether complete loss of SynGAP results in similar phenotypes as have been seen in mouse neurons. So to do this we use a system called MEA or multi-electrode array where we basically plated the neurons that are completely deficient for SynGAP or wild-type control neurons on a plate where we have multiple electrodes that can record spontaneous activity. So shown here in Panel A are just some of the neurons from one of the Syngap mutant lines that are plated on these electrodes. You can see here in Panel B recordings from neurons that completely lack SynGAP or that are wild type or have wild type sequence for Syngap. And then shown at the bottom here are individual recordings from the SynGAP deficient neurons and wild type controls. And I think what's really easy to see here these black lines indicate individual firing events. You can see in the neurons labeled homo1 and homo2 that there's a significant increase in the firing rate of neurons deficient for SynGAP compared to wild type control shown at the bottom. And that's true both at the level of individual neurons as well as in groups of neurons. So the pink lines here indicate that groups of neurons are firing altogether at the same time and what you can see is that in the SynGAP deficient neurons we have a lot of neurons that are firing together in both types of deficient neurons. You see a lot of firing in both of these neurons compared to wild type controls where we rarely see this coordinated firing at this age of neurons.

So again this indicates that complete loss of SynGAP results in excessive firing and excessive excitability of neurons compared to two wild type neurons. So why is this? So Gavin's group looked at this in a bit more detail and asked whether there might be a difference in the morphology of the neurons deficient for SynGAP compared to wild-type controls and found that those neurons that are deficient for SynGAP are significantly larger than the wild type controls and this is primarily and this is due to the fact that extensions from the neurons which are called neurites these are the processes through which neurons communicate with each other are significantly longer in the knockout neurons compared to the wild type neurons and we think that it's this excess excessive growth of these neurites of the dendrite of these dendrites of these neurons that leads to increased connectivity between neurons and the excessive firing that we see in the knockout neurons compared to the wild type neurons. So we've started to look at patient-derived neurons. We began while we were in the process of making these isogenic controls from our SYNGAP1 patients we decided to go ahead and do some recording from a SYNGAP1 patient and compare it to a neurotypical individual which is S3-084 here and then from these neurons that completely lack SynGAP which is Knock Out 38 here and what you can see in this very preliminary data is that the patient-derived neurons S3-059 have increased firing compared to those neurons from a neurotypical individual but not to the extent of neurons completely deficient for SYNGAP1. So again this is work that's ongoing now that it appears we have the isogenic controls we're going to begin looking at this in more detail in our patient line versus the corrected lines and we're also beginning to perform the same sort of gene editing in additional patient lines so that we can determine the severity of excessive firing that we see in different patient lines.

Okay. So I'm going to end with just a few slides about different modes of targeted therapies for neurodevelopmental disorders and I will say I'm going to go over just a couple of of types of therapies that I think might be particularly important for families that of children with SYNGAP1 that they've been thinking about or heard about
but this is an area that is really exciting to me because when I started my training in neurology about 10 years ago or so there really was nothing available for any neurodevelopmental disorder and just in the last five years a number of either gene replacement therapies or antisense oligonucleotides have become available for a number of neurodevelopmental disorders and I think this is something that is just going to continue to grow for the kids that I see. So I'll really focus on just a couple of types of therapy that might be pertinent for children with SYNGAP1 and that is in vivo gene replacement which is typically viral based as well as antisense oligonucleotides.

So gene replacement therapy as it's commonly studied these days uses a particular type of virus called adeno-associated virus and the reason this is used is because this virus tends to not elicit a strong immune response. It's something that unlike other viruses that have been used in the past such as adenovirus which does elicit a fairly strong immune response that can be detrimental to the individual that receives it. AAV does not have this issue. The other thing about AAV is that it can be administered in a number of ways.

So one way is directly into the brain which is called intraparenchymal. So this is a method where small holes, spur holes are drilled into the skull and then viruses administered directly into the brain. The virus can dissipate from where it's injected somewhat although this is one of the limitations is that it does not tend to infect all of the cells in the brain. AAV can also be administered into the ventricles or the fluid-filled spaces within the brain. This allows for the virus to circulate throughout the brain, so potentially can interact with more regions of the brain. However it only interacts with those regions that are directly bathed in this fluid. Again, one of the limitations of this approach. For certain disorders, for neuromuscular disorders an infection within the muscle might be beneficial and then for a particular type of AAV intravascular administration might be beneficial although there are some some caveats to that. So gene replacement therapy for neurodevelopmental disorders really came of age with this paper but in 2017 which was the first clinical trial that really definitively showed that a gene replacement strategy could be beneficial for a neurodevelopment disorder. In this case Spinal Muscular Atrophy. So shown here on the left is the first three patients that were administered an AAV that expressed the gene that's mutated in SMA which is SMN1 and then on the right a higher dose was administered to a much larger cohort of individuals and what you can see is basically most children with SMA on this score which is called the CHOP INTEND score score lower than 40. And what that means is they're basically not developing gross motor milestones normally. Kids with SMA, the natural history of the disorder is that these children with the most severe type never sit up, never roll over and typically pass away at a very early age. What was so exciting about this study is that nearly every child that was administered the higher dose of this gene replacement therapy did much better than the natural history studies would indicate that they should have done. And so this was very exciting because first example of a neurodevelopmental disorder that could be treated using this approach.

However there are some caveats with this sort of therapy. So one of them is that if an individual has existing immunity to the type of AAV that's being administered, the immune system will prevent that virus from infecting its target cells. They'll basically sequester it away. This is the natural immune response that we all develop to viruses. So for example either if someone becomes infected for example with the flu or if someone is immunized against measles this is the immune response that develops that prevents a second infection. So in individuals that have an established immune response to the particular AAV that's being developed for a given neurodevelopmental disorder, if that immune response is already present in the child that AAV will not infect the target cells instead will be sequestered away through the immune system. And as you can see in this graph especially if you look at children over eight years of age for most of the types of AAV most kids develop, over half of the kids in the study develop immunity to most of the AAV serotypes.

So this although this is very promising this might be something that is most promising for very young children and children over the age of eight or so are likely to have been been exposed to many of the stereotypes of AAV and this is potentially not quite as useful for those children. The other issue again that I mentioned before is that getting the AAV to infect every cell within the brain. So it really depends on the disorder whether this is important or not. For SYNGAP1 this is likely to be very important and the reason is most if not nearly every neuron within the brain expresses SynGAP and it's important that these neurons express the right amount of SynGAP. So having just 50 percent less SynGAP than normal results in the disorder the SYNGAP1 intellectual disability disorder. We also know that having too much at least in cultured neurons is detrimental to the function of those neurons. We don't know yet in model organisms or or in humans whether that is true as well. So it's important to get every neuron to express the right amount of SynGAP. Most of the... well all of the AAVs that are used currently are being investigated as a gene therapy vehicle are not able to infect every neuron. Even if the virus is placed directly into the brain it's still placed into only a few spots within the brain and diffuses only a short distance away. Those AAVs that can be administered for example systemically through the blood and they can cross the blood-brain barrier those only infect about one percent, one to two percent of the neurons in the brain. So again this is a limitation of the current AAV technologies. Something that's being worked on but something to keep in mind in terms of development of gene therapy, gene replacement therapies for neurodevelopmental disorders. The other thing is that it really seems for most neurodevelopmental disorders that age matters. That is the age at which the therapy is administered has a significant impact on the improvement of phenotypes. So this is just one study that looked at that for a disorder called mucopolysaccharidosis. A gene replacement therapy was administered intraparenchymally into four children of various ages from about 20 months of age to a little over 50 months of age and what you can see in the small clinical trial is that the child that's administered the earliest at 20 months of age seemed to do the best. Their developmental trajectory actually paralleled that of neurotypical child. Whereas the child that was administered to therapy the latest at 50 months they did see some improvement in terms of their development, in terms of not regressing to the same degree as was seen in the natural history study but still not in line with the development of neurotypical kids.

Okay and then I'm going to talk about antisense oligonucleotides. This is again something that's very promising for a number of neurodevelopmental disorders and I think something that certainly a lot of my families talk to me about whether this is something that could be used for kids with SynGAP and I think there is a lot of promise for this. So just to kind of orient you to molecular biology so all genes are encoded by DNA including SYNGAP1. This DNA is translated into mRNA this is sort of an intermediate step leading to the final product which is the SynGAP protein. Antisense oligonucleotides as they were originally developed were a method for decreasing expression of a protein. So shown here is a typical mRNA. When an antisense oligonucleotide binds to this mRNA, binds to the middle of this mRNA it's degraded and this results in a reduction in protein production. Obviously this is not what you want for SYNGAP1 but antisense oligonucleotides can work in different ways depending on how they're developed. So again the sort of classical way is to decrease expression through mRNA degradation but it's also possible to alter which exons are included in the final transcript product and therefore the type of protein that's made or for SYNGAP1 what's most promising is that you can target other parts of the mRNA or other molecules to actually increase the abundance of a given protein.

So one way is to sequester something called microRNAs that normally regulate protein production but other things that can be targeted include things called natural antisense transcripts or poison exons. These are regulatory mechanisms to the cells normally have, neurons normally have, to precisely regulate how much protein is present. In an individual with a loss of function mutation in Syngap it would be beneficial to increase the amount of protein. So using antisense oligonucleotides to target antisense transcripts which normally decrease the amount of SYNGAP1 could lead to an increase in its overall abundance or as a recent paper showed targeting what are called poison exons which also lead to degradation of mRNA and prevent protein translation. Targeting those poison exons could also potentially increase SynGAP abundance. And then I just wanted to show the one example or probably the best known example of an antisense oligonucleotide for neurodevelopmental disorder again for SMA where in this case they're not trying to decrease the amount of a protein but they're actually trying to increase the amount of a certain protein product and to do this they use an antisense oligonucleotide that alters the protein that's made from a related gene to the gene that causes SMA which is SMN2 and results in what is nearly a normal protein product from this copy of SMN1. ASOs can be administered also in a number of ways. They can be administered directly into the brain They can be administered through a spinal tap. There's some work going on to look at whether they can be administered intranasally or systemically. For SYNGAP1 because the most prominent phenotype is cognitive and seizures it's likely the intraventricular administration would be necessary and I just want to give you an example of how this would potentially be done.

This is shows you a medication that is now used for a disorder called ceroid lipofuscinoses. This is not an ASO. It's an enzyme product. Nevertheless this is potentially a similar mechanism would be used in individuals with a cognitive neurodevelopmental disorder where a port is placed directly into the ventricle of a brain and then a biological substance could be administered either an enzyme in this case or an ASO for other neurodevelopmental disorders. So this is, you know, obviously this is very exciting because children with this particular disorder have significant improvement in their development but there are also challenges with this. Having a port that's permanently connected directly to your brain can result in infections from the outside world getting into the brain and significant hospitalizations and illnesses associated with it. So with all of these while there's a lot of promise there are also potential challenges that need to be overcome. Okay. So I think I may have gone over just a couple of minutes. So I'll just quickly say that, you know, what we've been looking for and what I talked about today or trying to understand what clinical endpoints or biomarkers might be meaningful for clinical trials certainly sensory perception appears to be one that would be very important to look at. Sleep abnormalities could be a very important endpoint. Seizure activity, EEG, particularly EEG findings such as these posterior generalized discharges could be important. Behavioral outbursts and other behavioral abnormalities and of course developmental delay and intellectual disability, cognitive outcomes and then for SYNGAP1, you know, I think we can't rule out using things like small molecule modulators for example for SynGAP protein stability. Something that Gavin's been working on for a number of years. Our lab is also looking at genetic modifiers of SynGAP protein stability as potential entry points. ASOs have a lot of promise although some of the caveats I mentioned. Still I think the jury's out about gene replacement technologies. I think we need new technologies in that regard and then I'm asked very frequently about stem cells. At this point there are... it's not clear evidence that stem cell transplantation would have any impact in children with SYNGAP1. I think there needs to be a lot more research in this regard.

So I want to end by thanking all the families that are on the call, all of the families that have participated in our research studies. You guys are what drive me every day and also of course all the funding sources especially the SynGAP Research Fund have been very generous and helped us with launched the neuron work that we've been doing in the lab. So I will end there and I went over so I don't know if there's time for questions or not but I'm happy to stick around.

Mike: That's very kind of you. Thank you and thank you for the presentation. We lost a few people at the bottom of the hour but there's a few families on. If you are a parent or an SRF member who wants to talk I can put you up on the panel. I'm happy to do that. First, Dr Holder thanks. That was quite a tour de force and a great overview. I think the thing about the age is going to bum people out and I think the thing about the needle in the brain maybe it's just recency because it's the last slide I just saw but there's a bit of a head twister too because I think people were getting their head wrapped around intrathecal for ASO. So I would have a question on both of them. Why do you think it would be that versus intrathecal? Just to ensure widespread delivery? And then on the age thing I'm sure you're following this too, the recent Angel- FAST, the Angelman organization just published some pretty breathless initial readouts from the docs about wow we didn't think... I mean the one that stuck in my head was "we didn't think the brain was this plastic" this is remarkable. So I totally understand as a clinician who's got desperate and tired parents like myself coming into your lab your job is to be clear-eyed with us but is there also room to say there's still stuff we don't know? Not to put words in your mouth but just to stay balanced?

Dr Holder: Oh there... absolutely. That is absolutely true. So you know I just, I wanted to put that out there as something that is probably a factor but it's... if I made it seem like, you know, after a certain age there's no hope I did not mean to do that. I think it plays a factor. That there is a, you know, an age is a factor in the efficacy of these therapies but from animal models and, you know, from some very preliminary data as you were talking about from patients what's clear is the brain is very plastic and there is room for improvement and that's why you know when when I talk about therapies you notice I never use the word cure right? I'm not trying to say I'm going to develop the cure and everyone just wait, you know, it's coming. I think what we need and what we can achieve in the near future are therapies that improve quality of life, improve symptoms for our patients with SYNGAP1 as well as for other neurodevelopmental disorders. That's what I'm working towards and while age is a factor that's just one factor. There are many other factors. I think potentially almost equally as important is combining these personalized therapies with traditional therapies with the speech therapy, with physical therapy, with fine motor therapy, with ABA therapy it's that's going to be really critical is not to expect that there's going to be a pill that's going to reverse everything, to really get a comprehensive therapy.

Mike: Thanks for that. Can you see the Q&A? There's a few questions that came up. JR has been prolific as usual and then Rebecca has some questions and it looks like we have one person who's got a question about one of your papers from their class.

Dr Holder: Okay. Let me read these. So to Dr Holder how do you suggest identifying doctors in a variety of regions educated and ready to focus on Syngap patients? Pediatric and adult? Super hard and I'm getting referrals still from all over the country and calls you know, "I have this kid with a Syngap mutation what do I do?" and those are you know very, very frequent. Certainly I'm not sure where you are exactly but the larger medical centers are more likely to have individuals that are somewhat versed in these rare neurogenetic disorders but you know it's Syngap mutations were again only identified 11 years ago. So it's a difficult... it's very difficult and I can tell you in Houston what to do but it's harder around the country. I fully realize that. Second question... oh sorry go ahead?

Mike: I just want to pile on to that because it is something that you know, as a patient advocacy group we get the newly diagnosed parents and some of them are next to big centers like your own and I say go find a neuro who's if they haven't seen a Syngap look for someone who's seen Dravet or Angelman or some big disease because at least they have a clue as what the genetic epilepsy might look like. Sure. But when I talk to people who are in the more remote areas that don't have access to great you know cadres of neurologists I urge them to travel but we're going to see more and more kids and we're going to see more and more adults. Right? We're already seeing a handful of adults in fact we just found a 65 year old like a month ago we were making a movie about. How do you as one of the... and you I mean if I think about like who knows which clinicians are leading clinician-scientists are leading the charge on thinking about how to address Syngap in patients in clinic, it's it's really you and Dr Connie top the list along with others like Dr Poduri but as there's an explosion of diagnosed patients what is the way the medical industry works to take the knowledge from you and Dr Connie and share that? Is it just you publishing papers? Is it a seminar? Is it continuing at AES? Like, because we were expecting a bit of a hockey stick but how do we catch the clinicians up?

Dr Holder: Yeah, so that's a great question. I certainly try to do my part to educate through those platforms and Connie does as well. Part of it is you know, the younger generations of physicians are just becoming more versed in these neurogenetic disorders and they're becoming more comfortable with, you know, reading about them and trying to understand them. It's a steep road though. I mean even something that's been around for a long time that, you know, like Rett syndrome that more people know about, you know, depending on your practice you may only have one child with Rett syndrome and so you just don't have the expertise. I will say Connie and I, I can speak for myself and I think I can speak for Connie as well we're always happy to speak to your local neurologist and I do this fairly frequently. So that's another way. Certainly, you know, advocacy organizations putting information out there is really important as well.

Mike: Okay do you want to go back to the questions?

Dr Holder: Sure. So the next one is how can we find research-based clinicians to study other potential aspects of syngap disorder? Pituitary and enteric nervous system and bladder control sensation? That's... yeah, that's a great question. So I can tell you for the other disorder I study Phelan-Mcdermid syndrome which was first described in the 1980s right, so you're talking about 20, 30 years. For that disorder there only now are coming online GI specialists, endocrinologists that are really studying those aspects of that disorder and so certainly again because Syngap is identified so recently as a cause of neurodevelopment disorder we don't have that yet. As far as I know. We do... we have some interest in GI issues more on the basic science side with Julia Dallman down at University of Miami. Someone that worked with me here at Texas Children's is now in Florida and is helping her kind of more on the clinical side with GI issues. So that's kind of what I'm doing. I'm trying to teach the trainees here and then they go off to the world and hopefully we can just build it that way.

Mike: And then Rebecca's next question is are you hoping the data from the gait studies could potentially identify undiagnosed kids? This would be a holy grail right? Some way of seeing if someone's Syngapian but without having to go through a genetic panel.

Dr Holder: Yeah so I think the gait study is probably not going to be specific enough to identify whether a child has a SYNGAP1 mutation versus another mutation in a different neurodevelopmental gene. I'm optimistic. I'm hopeful that you know, I understand the issues with with genetic studies in terms of getting insurance companies to pay for them, in terms of their availability. I'm hoping that that will more and more be available. So for example, an older technology that's still used and diagnosed a lot of kids with Phelan Mcdermid syndrome are chromosome microarrays and as of five years ago it's really hard to get an insurance company to pay for those. Those are now nearly universally paid for by insurance companies. I think exome sequencing will go that way. It's just it takes time because often insurance companies they just put up barriers to try to reduce their costs. You have to prove to them that it's important to get the specific diagnosis and that will still reduce their cost because now you have a better idea of what to focus on. So you kind of have to sell them through the literature basically that it's important to do that so.

Mike: Then I'll let you read Rebecca's next question.

Dr Holder: Mmm okay. So using the gait studies will you correlate with imaging studies that may have identified orthopedic problems in patients to identify prevalence of potential issues for future patients to be aware of? Great question. Not something that we're looking at in this first group of patients. We really just want to define what are the gait abnormalities but then obviously the next question is you know why do these gait abnormalities occur? Is it because of neurologic dysfunction? Is it because of orthopedic issues? That's a good question. Anonymous sends in: in one of my classes this semester we discussed your 2019 paper where you re-express SynGAP in adult mice and found improvements brain function, behavior is curious of theory and progress along this line of treatment? So I will say this is Gavin Rumbaugh's paper. I mean we contributed but it's really his work and so from that paper really you know spraying the work that we're doing now to try to identify genetic modifiers that when inhibited increase SynGAP abundance. So that's kind of the next step from my lab. For the Rumbaugh lab he's looking at compounds that might, small molecule compounds that might increase SynGAP abundance. So yes there is ongoing work related to that study.

Mike: Okay and then JR, Virginie, Rebecca, Virginie and you're off the hook.

Marta: Okay I have a question about the EEGs. You know it has come up several times also with Dr Hicks. Do you think the EEGs are gonna be specific enough to have a biomarker on the EEGs? Because it keeps coming up and I think do you have the interest, Doctor Hicks has the interest. Do you think if it's studied properly we get the biomarker from the EEGs?

Dr Holder: Yeah so I mean there are a lot of different ways you can use EEGs potentially as a biomarker. I talked about some of the common abnormalities that we see in kids with SYNGAP1 mutations. For a another, a different clinical trial that we're doing related to Phelan Mcdermid syndrome we're looking at the frequency of epileptiform discharges. These are these abnormal discharges not associated with seizure but indicative of irritability, you know, that can lead to a seizure. So one thing you can do is you can just look at the frequency of those discharges either in a given individual before and after treatment and see if there's improvement with whatever treatment you're using in combination with other things like seizure frequency. So I think EEGs can be used as a biomarker. I don't think... I'm not sure there's one biomarker, there's one EEG abnormality that will universally work as a biomarker for every individual with Syngap. For example although that posterior intermittent rhythmic delta activity is very common in kids with Syngap mutations it's not universal and it's probably present in 50 to 60 percent of kids with Syngap mutations. So it's, you know, it's frequent enough it could be used but it's not universal. What's more common are these epileptiform discharges and you could look at the frequency of those and see if there's a change but again that- any sort of biomarker has to be coupled with a clinical outcome measure that improves. So you- a clinical trial cannot look just at a biomarker has to, you have to show that there's some improvement in the phenotype that will be clinically meaningful for that child.

Marta: And another question we I mean we get like in the groups frequently as is in a child I mean being Syngap considered epileptic encephalopathy, if a child has abnormal EEG but doesn't have clinical seizures do you put the do you because some clinicians don't put those childs in anti-epileptic medications and some do. What is your take on that? We get that question sometimes and it's kind of all over the place.

Dr Holder: Sure. Sure. So one thing is that some of the children that have those abnormal discharges but do not have clinical seizures may develop the clinical seizures in the future depending on their age. But I have definitely seen some kids with Syngap mutations who are older, who are teenagers and have not had any clinical seizures. So for those kids even if they have abnormalities on the their EEG I do not put them on medication. The reason being I don't know how to titrate the medication. So for seizure activity I try to titrate a medicine until there's no seizure activity but for EEG abnormalities most of the time those abnormalities do not disappear with medication. Sometimes they do but most of the time they don't. So then I don't really have an endpoint to which to titrate that medicine to. I think what, you know, still is sort of out for debate and more work needs to be done is whether those EEG abnormalities lead to some of the other phenotypes that we see other than seizure. Do they cause some of the behavioral problems that we see? Do they cause... are they indicative of the severity of intellectual disability or developmental delay? And that's really a research question. So that that's something that needs to be addressed not just for Syngap but for many neurodevelopmental disorders.

Mike: All right. So the next question was can you speak to how we might learn about the Angelman syndrome ASO trial? Would that disease be similar to Syngap as far as affecting every cell in the brain?

Dr Holder: So... yes and so it's a complicated question actually because it's an imprinted gene but for all intents and purposes, yes it's every region of the brain is affected in Angelman syndrome similar to Syngap.

Mike: Can I actually stop you there? Sure I you.. I realize that it's...we're getting heady here but the Angelman people have a social media machine that I am envious of and everyone knows exactly what's going on in Angelman but that the difference in that whereas we have a variety of mutations in there and imprinted can you just double click once on kind of where it's important to learn from them but we're a little bit different than their disease and this is my editorializing as I do. You can, you're free to disagree or not maybe Dravet a is a better analog and we should be watching them which is something that just in terms of a classic haploinsufficiency, variety of mutations, but Angelman and Dravet get thrown around a lot and if you could just dwell up on that in a minute?

Dr Holder: Okay.

Mike: I'd appreciate it.

Dr Holder: Yeah. Yeah so you know both Angelman and Dravet syndrome have a lot of similarities with Syngap in terms of phenotypes, in terms of cognitive development issues, in terms of epilepsy being very frequent in both of those disorders. So in that sense Syngap is very similar. When you drill down to the specific genetic causes of these disorders they're all very different. So Angelman syndrome is primarily due to a mutation in a gene that regulates other proteins. So the gene creates a protein that regulates other proteins. It does so in a different way than SynGAP. So SynGAP does the same thing. It regulates other proteins but only a very small, very specific type of protein called a G protein. Whereas the the gene that we think is primarily responsible for Angelman syndrome, it regulates the stability of other proteins. The genetics are a bit different in that for Syngap the mutations in general probably 90, 80-90% of the mutations are loss of function mutations, point mutation in the gene. So in the end you have 50% reduction of the protein the normal amount of protein that you need. For Angelman syndrome there are a variety of ways that it can occur. You can have point mutations. That that is one way that you can get Angelman syndrome point mutations in the gene called UBE3A can result in the Angelman phenotype. You can have a deletion of the region in which UBE3A is present and that can also result in Angelman syndrome and then you can have abnormalities in imprinting and really it's in the issue here is that you can get what's called diparental parental disomy where instead of getting one copy from mom and one from dad, you get two copies from one parent and that results in the phenotype because of imprinting. For Dravet syndrome there many of the mutations are actually missense mutations that alter the the function of the protein and actually for Dravet the other difference is that particular protein seems to be most important in inhibitory neurons and those neurons become less functional which results in the increased excitation of the brain. Whereas in Syngap it seems to be that the protein... there's some debate here but the protein is most important in excitatory neurons and this function or loss of the normal abundance of SynGAP results in hyper excitation of those excitatory neurons because of an intrinsic dysfunction in those in those neurons. If you had a panel of scientists here they might debate me right and talk about how interneurons are actually really important but I'm going to give you kind of what is generally thought to be the case.

Mike: I appreciate it. I take your point. I mean I'm not saying SCN1A and SYNGAP1 are the same but sort of the classic neuro haploinsufficiency versus Angelman which is a little bit I try to tell people don't assume that we can do what we did in Angelman in Syngap. Like I mean you had on your list the bone marrow transplant. That's a whole other crazy town Angelman thing. Right and there are disorders, there are disorders, neurodevelopmental disorders in children where bone marrow transplantation is the is the clinical standard. So just depends on the disorder which is why we're lucky to have you and others working on our disorder. Okay let's let's keep cruising here. So Rebecca had a question do you see potential benefits of what you've seen nice question Rebecca do you see potential benefits what you've seen of Ciitizen and helping you with so Ciitizen to remind you is we're going to have 100 patients normalizing all their data in one place in an effort to find more of the that fabulous charts you had sort of showing how our kids show up.

Dr Holder: Yeah so I think it's critically important for a number of reasons one is that there's never going to be one center that sees 100 kids or at least not in the near future in the term in the time span the citizen will be able to aggregate that number of records. So that's really important as you saw from some of what I presented you know you can begin to look at things like medication efficacy and then I know with Ciitizen in particular using machine learning you might be able to make connections that you know the human brain would not necessarily go to. This medication that we use for seizures improves gait for example, something like that and that's what we're using that sort of platform that utilizes machine learning can be very beneficial. You know there are everything has its place and I will say that's not going to replace prospective natural history studies because those use different tools to measure things in a certain way whereas retrospective analysis, you know, you can get more records but then the records are more heterogeneous. Right? So everything has its place and you know I definitely think it's you know the the effort's very important but I want to encourage people not to say well I'm participating in Ciitizens so Dr Holder's questionnaires are useless.

Mike: I have no no no I would say or sign up for Ciitizens only takes 10 minutes and then you have all your medical records and then go and do all Holders questionnaires because you're going to remember stuff because I've only been diagnosed two and a half years I can't remember a year ago and it is an exciting discussion around these debates and I'm not for a second because you and Ingrid both are quick to correct me on you know the gold standard is a classic natural history study and the reminder I reply with is yeah but those take years and cost millions whereas Ciitizen you know we collected data three months ago people signed up three months ago and Ellie shared a first readout of only 10 but you know we're up to 75 so the number keeps growing last week so the speed if it can help us at all is I think welcome and it's not costing us millions. Thank you very much for for sharing this and for the update. This is a recording we're going to send a lot of new families to go and watch the existing ones as well and we will follow up probably with a link ask you for a link or how people should reach out if they want to participate in these studies that they can. Okay all right thank you so much. Thanks everybody for joining.

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