AADCd Family Workshop 5; Understanding the Biochemistry in AADCd ENGLISH

AADCd Family Workshop 5: Transcript Understanding the Biochemistry in AADCd

Professor Simon Heales UK

Transcript ...

Lisa

Hello everyone, I will start as we have all week, so sorry for the repetition for those of you who have heard this all before.

Welcome Professor Heales, thank you all for joining today. My name is Lisa Flint, I am the founder of The AADC Research Trust and mum to 23 year old Jake who has AADCd. Together with my colleague Julie and all of you we are celebrating 15 YEARS this year of patient advocacy, raising global awareness and funding critical disease research.

The subject of Biochemistry in AADC deficiency is of extremely high interest and we hope you find this final workshop really helpful.

Let's begin by welcoming and introducing our presenter. Professor Simon Heales has been scientifically working in the field on neurotransmitters for over thirty years. He was part of the original team who diagnosed the very first ever children in the world with AADC deficiency in 1989. Then subsequently went on to diagnose the first British child, seventeenth in the world, which was my son Jake.

From then on Professor Heales has been a firm family friend, a fantastic AADC global advocate, a co-founder and director of The AADC Trust, a dedicated disease researcher and a staunch supporter of all of our children.

Simon Heales is a Professor of clinical chemistry at University College London, Head of the Neurometabolic Unit at National Hospital London and also Head of Enzyme and Metabolic unit at the world-renowned Great Ormond Street Children's Hospital also in London.

Welcome Simon and we look forward to your presentation.

Professor Simon Heales Thank you very much for that introduction Lisa. It's an absolute pleasure to talk to you all today regarding the biochemistry of AADC deficiency. I really hope I can demystify some of the biochemistry, explain some of the results you may get and answer some of the questions you may have around this inherited metabolic disease.

So I hope not to be too heavy duty biochemistry on a Friday afternoon, here in Page 1

London, and that you can understand the full process whereby we make a diagnosis. Hopefully I will be able to explain to you the consequences of the enzyme deficiency that we are talking about, Aromatic Amino Acid Decarboxylase.

Slide 1 When we are thinking about AADC we are thinking about a family of neurotransmitter disorders. Whenever we think about neurotransmitter disorders we need to think about neurotransmission. A way of thinking about neurotransmission is as communication.

So we have these funny looking cells here, these are the ends of our nerve cells and they are trying to talk to each other. Those white dots coming from the one above to the one below, this is going on in our brains, those white dots are whereby the funny looking cell at the top is communicating with the cell below.

So it's saying hello, and it's saying hello by releasing a neurotransmitter. Those are the white dots coming from the top to the bottom.

This is how our nerve cells communicate with each other. These nerve cells run through our brains through our bodies, into our muscles, etc. It controls so many processes, for example movement, and one of the key neurotransmitters involved in movement is one that we have all heard of called dopamine.

So dopamine can be some of those white dots that are enabling cells to communicate with each other. I think we all know in this world how important communication is. If we don't communicate things don't go as well as they should.

And in a neurotransmitter disorder we have a degree of breakdown of communication, so then the neurotransmitters are not being made in sufficient quantities and the communication between those cell types breaks down.

So if it’s a dopamine molecule you’re going to have less dopamine, in a deficient state, such as AADC deficiency, and that's going to impair, for example, the ability to have very fine controlled movement.

Another communicating molecule that we are all familiar with, is serotonin and that again is doing a similar job to dopamine - communication. That can affect mood, digestion, etc, that's a communicating molecule that's involved in those types of processes. So what we are going to be concentrating on are the communicating molecules dopamine and serotonin.

That's what I mean by neurotransmitters and this is a process of neurotransmission. Biochemistry is a fascinating subject and if we are not careful Page 2

we can put people off it because it always looks a bit too complicated.

Slide 2 Here we have a metabolic biochemistry pathway and this is just a snapshot of some of the chemical reactions that are going on in every single cell in our body.

I hope you will forgive me but this afternoon I am not going to go through every step here in this metabolic pathway. One, because it's not necessary and two, I don't know everything about it but we just want to drill down on the dopamine and serotonin pathway.

I for one actually like these maps which are like road maps, as you can see the journeys of these chemicals going along these pathways to allow these key chemical reactions to occur. I think we all agree this is far too complicated at any point, let alone on a Friday afternoon to dig deep here so we are going to start to move further down into the neurotransmitter pathway, in particular the neurotransmitters dopamine and serotonin.

Slide 3 The next slide is slightly less terrifying now, but still a bit complicated, this is showing us the black arrows are the enzymes, the squares are the names of the enzymes and in yellow are the chemicals that we are interested in. That's still a little heavy duty with regards to how we are going to talk about neurotransmission. Certainly in our laboratory we are not referring to pathways like this.

Slide 4 We go a step further down and this is the pathway that I will be using. So I have got from that complicated pathway, getting simpler and simpler, to really focus now on the pathways that we are interested in. And we are interested in dopamine and serotonin and you can see these on the slides that I am showing.

So we are going to move left to right. That's the journey of our traffic today.

That's the biochemical reactions that we are going to consider and explain, I hope, dopamine and serotonin production. I also want to explain to you what we measure in the laboratory to help achieve a diagnosis of a dopamine or serotonin deficiency state.

So on the left hand side, we are going to look at tyrosine first of all. Tyrosine is an amino acid and you may or may not know that amino acids are the building blocks of proteins. They get bigger and bigger these amino acids and they make Page 3

lots of really key important proteins. But on their own a single amino acid can be involved in a metabolic pathway. For us tyrosine is a key molecule.

So now we are going to move from tyrosine through the blue arrow to L-Dopa. It's a word change and tyrosine has now gone to L-Dopa. The blue arrow represents an enzyme. Enzymes make reactions occur. If we just had some tyrosine sitting on the laboratory bench, it would take many many years for it to naturally progress to form L-Dopa, it wouldn't happen naturally.

So what enzymes do is they make chemical reactions happen. They make the impossible possible. So when this enzyme is present tyrosine hydroxylase as we call it, takes hold of the tyrosine and makes the next step in the pathway which is L-Dopa.

So we've heard of the enzyme tyrosine hydroxylase making L-Dopa, but that's not the final neurotransmitter. The neurotransmitter that we want is dopamine.

L-Dopa is a precursor. It's not active, it can't do the communicating job, but there's another enzyme, another reaction that is facilitated by the enzyme AADC or Aromatic Amino Acid Decarboxylase. That takes the L-Dopa molecule and transforms it into dopamine. So you can see in blue that's another enzymatic step and we have now made the molecule dopamine.

So tyrosine to L-Dopa, tyrosine hydroxylase an enzyme, L-Dopa to dopamine, our well known enzyme AADC has now made the dopamine molecule. That is now available to the cell to do the role. It can now be released in a controlled way and work on target cells to cause a response. So with dopamine it might be to control movement.

We don't measure in our laboratory dopamine itself. Dopamine has a waste product. So when it's done its job, it binds its the receptors, it does the job in the target cell, then other enzymes are around to break it down. In our bodies a group of enzymes, there's a blue arrow again, breaks the dopamine down to it's waste product and that's known as HVA or homovanillic acid but in the lab we never call it that. I go into the lab every day and say what was the HVA level like?

So if you look at that pathway in red, HVA is what we measure in the spinal fluid, which we will come on to a bit later, the cerebral spinal fluid the CSF. The HVA is our marker of the overall integrity on how well tyrosine is working through these steps to give you dopamine and that's why we measure the HVA. If we have an enzyme deficiency which we are coming to a bit later, if the block was here it's HVA, if the enzyme here was missing tyrosine hydroxylase that would Page 4

also impair the ability of the brain and other cells to make dopamine. So the HVA would be low. So hopefully we have cracked dopamine production.

Now let's move to the next level down and let's talk about this other neurotransmitter serotonin. Serotonin is a key communicating molecule. This time you replace tyrosine with tryptophan, a different amino acid. And a different enzyme tryptophan hydroxylase makes 5-hydroxytryptophan (5HTP). So we've gone from a tryptophan molecule, it's been chemically modified by the enzyme and it's now become one step away from making serotonin. The exact same enzyme that took L-Dopa to dopamine takes 5-hydroxytrytophan and converts it to serotonin.

So now you've got this key role for the AADC enzyme. It is absolutely essential that this enzyme works to make adequate supplies of dopamine and adequate supplies of serotonin. That's our focus, so if there's an enzyme deficiency at that level you then have a deficiency after the enzyme of dopamine and serotonin.

We don't measure serotonin directly in our laboratory what we measure is the waste product, just like HVA reflects dopamine, 5HIAA, it's in red because we measure it and it's a marker for the overall integrity of the pathway to take tryptophan all the way through to serotonin.

Hopefully on this pathway I have given you and orientated you on the AADC enzyme and how the brain makes dopamine and serotonin. And hopefully begun to identify the consequences of when that enzyme is missing.

Underneath I have written PLP, that is pyridoxal phosphate, it's the active form of Vitamin B6. Some of you may have heard of Vitamin B6, sometimes it is considered as one of the treatment options to consider and that's because the AADC enzyme to work maximally and to be really working well, needs pyridoxal phosphate under normal conditions. So it's possible for some mutations that the AADC enzyme even though it's very low you may get a little boost if you give Vitamin B6. It's not fully proven yet but there is a logic there to giving pyridoxal phosphate to boost any remaining enzyme activity. So that's why you hear about Vitamin B6 because it's key role, it's called a co factor. A co-factor works with the enzyme the two are best friends, they have to work together, one doesn't work on its own, you have to have the enzyme and the co factor to allow the biochemical reaction to proceed.

Slide 5 If we think about the consequences of those lack of communication molecules you can start to see the problems that can arise, because we know very well what these neurotransmitters, these communication molecules do.

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On this slide I've got the consequences of dopamine deficiency and that's linked to that key role dopamine plays throughout our bodies and what happens as a consequence of the deficiency of dopamine. And equally I've got serotonin as well. You can see also the consequences because this is linked to what you no longer have. The serotonin is not being produced sufficiently so the consequence of that is you get these problems listed here.

Slide 6 Let's just go back to this slide a second more. If you look at dopamine there, there's some more steps that I haven't included, but the next step, dopamine has other options. It can stay as dopamine or it can get metabolised to noradrenaline/norepinephrine and norepinephrine can get further metabolised to epinephrine/adrenaline.

So you can see I can put more arrows in now and go back to my earlier slides and this more and more complicated. The important thing here is that dopamine is deficient in AADC deficiency. So if dopamine is deficient we see the low HVA but because dopamine can go on and make norepinephrine and epinephrine they also becomes at risk and can be decreased.

Serotonin is a precursor of melatonin. So we've got complications further down the pathways as a consequence of this enzyme deficiency.

Slide 7 So if we go back to this slide we've got the consequences of the dopamine deficiency, the consequences of the serotonin deficiency and then the bit on the slide that I haven't mentioned until now the consequences of the norepinephrine/noradrenaline and the epinephrine/adrenaline deficiency as well.

So we can put this together based on our knowledge of the neurotransmitters that are deficient in AADCd.

Slide 8 What we are talking about here is an inborn error of metabolism. I want to use this slide because hopefully as I progress in this presentation I will be able to explain to you the results we get in the laboratory based on our understanding of the concept of an inborn error of metabolism.

Here we have A going to B, B going to C, and C going to D. So that could be Page 6

tyrosine going to L-Dopa, L-Dopa going to dopamine, perhaps dopamine going on to norepinephrine/noradrenaline. It's a metabolic pathway. What I've been describing when I've said tryptophan to serotonin or tyrosine to dopamine are what we call metabolic pathways.

To get a reaction to occur you need the enzyme, ENZ in this slide, so that tyrosine hydroxylase or your AADC enzyme. Now remember I said they need a partner, a co factor. So for the tyrosine hydroxylase it's partner of choice is tetrahydrobiopterin, so you may sometimes hear about terrin disorders when we are talking about neurotransmitter disorders. For AADC it's pyridoxal phosphate, vitamin B6. So you can see what I mean, to get the enzyme to work you need that co factor.

So when you have an enzyme deficiency, I have created an enzyme deficiency here with a red arrow, look it's gone. D has gone down and C has built up. And that's what happens in a classic inborn error of metabolism. In AADC deficiency, before the enzyme, so the L-Dopa molecule, will build up and the dopamine will decrease because you've created a block and therefore the flow of traffic on this metabolic pathway has been markedly impaired. D has gone down and C has gone up. And upstream, B is going up and A is going up. So there we were before and now they've grown because they've got nowhere to go.

And the analogy that I have used many times is the motorway on a Friday night.

This is called the M25 in London, it's basically one of the world's biggest car parks because everyone on a Friday night, in non Covid times, is trying to rush home. There I've got all the cars going around there. All those cars are the chemicals, they're the amino acids, they're the neurotransmitters that are being produced, or not produced in this case, all the chemicals that you need and they are going along that motorway but unfortunately there's been a car crash further around on the motorway and that's created a block and now those cars have got nowhere to go. So we are getting a build up of anything upstream of that car crash. On the other side of the block there is hardly anything, so we just have just one single lorry coming along.

So it's about, in my mind, inherited disease can be seen as a failure of the flow of traffic. And so the traffic gets stopped and builds up earlier on before the car crash in this case, but afterwards there is a deficiency because nothing can get through. And actually what you're seeing is that the drivers are going off on another lane now and what sometimes happens in these diseases is that you open up new metabolic pathways that didn't exist before, because the traffic has got to go somewhere. And in some disorders it's not just the deficiency that's causing the disease it's the build up and opening of other pathways that can also be disease causing.

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In AADC deficiency what we are talking about is deficiency of the dopamine and serotonin so we are after the metabolic block but we mustn't lose sight that we are getting things building up before the block as well. In the laboratory this will help us to make a diagnosis. In the laboratory we are always looking at the metabolic pathway to dissect out where this block may occur.

Slide 9 Let's look at that pathway again. Hopefully it's becoming familiar with you. We've got tyrosine going to L-Dopa, in red we've got the AADC enzyme and we've got dopamine the other side and HVA that we measure. Now remember when I was saying about the motorway those drivers, the cars when building up they went along a different pathway. They opened up a new road to go down. So L-Dopa does the same thing. If you look at that block, AADC is the block, dopamine is deficient and L-Dopa is building up. The dotted line there is showing you that a new pathway is opening and that one there is 3-methyldopa. That is a metabolite of L-Dopa. We don't normally get very high levels of that but because we've got the block now we are building up the 3-methyldopa. We can measure that in dry blood spots. So there's the block, AADC deficiency, has built up 3-methyldopa and so we've now got an accumulation of 3-methyldopa, which can be measured in dry blood spots. So not only is it building up in the cerebral spinal fluid it is also building up, outside of the central nervous system and you can pick that up in dry blood spots. There is dry blood spot testing for elevated 3-methyldopa which can be an indicator as I've shown here of AADC deficiency.

Furthermore, 3-methyldopa can go on and be metabolised further into something called Vanilactic Acid or VLA and that appears in the urine. That can be picked up in something we call the urine organic acid profile. Urine outside of the brain but it's coming from the build up of L-Dopa.

So this dotted line is a new pathway that is opening up and giving you those metabolites, in this case in the blood, 3-methyldopa, and in the urine vanilactic acid.

There you can see a green arrow going into my blue dotted arrow and 5MTHF stands for 5-mehtyltetrahydropholate, so folates, which is a key vitamin. Why have I got it on there? Well if you look at L-Dopa it's going to 3-methyldopa, it's added the methyl bit to it. If you look at 5MTHF the 'M' stands for methyl. So what's happening is the 5-Methyltetrahydropholate gives it methyl to L-Dopa and make3-methyldopa. So if that pathway is particularly active you start to deplete the cells of folate, because the folate is being used to drive the reaction to create 3-methyldopa. So that's why in some cases of AADC deficiency, depending on how active that pathway is, that can influence your folate levels.

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So that's why we talk about the 5-methyltetrahydropholate status. Now if we were all together in a room I'd get you to do a quiz with me and I'd ask you all say higher or lower.

So do you think in AADC deficiency HVA and 5HIA are going to be high or low?

Low If we go upstream now, above the block, do you think L-Dopa and 5-hydroxytryptophan are going to be high or low?

High What about 3-methyldopa, is that going to be high or low?

High And vanilactic acid, high or low?

High What about 5-methyltetrahyropholate, high or low?

Low. But there is a dotted line because it's not always low, it depends on how active that pathway.

What I've tried to do here, this is my take home message, this is exactly how we work in the laboratory. The low levels and the high levels together that enable us to make a diagnosis of AADC deficiency.

As I've said 3-methyldopa can be done on dry blood spots. So where you don't have access to CSF testing all the time, it could be an initial investigation. It has to be followed up as 3-methyldopa can be elevated in other conditions as well, but it's a useful way in. And remember all the laboratory results are always done in context with the clinical details, so we very much work very closely with the clinicians. And valinatic acid can appear in the urine so we can look at the urine status as well. And these are very important as you put the whole case together whilst we are waiting for enzyme activity, for example, because you can measure the enzyme itself in blood plasma and you've also got access to genetics as well.

So it's the whole picture that we are putting together when we are investigating the patients. Of course I have a bias to the biochemistry because I am a biochemist, but it's everything together.

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Slide 10 So what we do in the laboratory is we look at CSF, so when investigating a potential case of AADC deficiency right at the start is the patient and often the parents, depending on the age of the child, invariably the parents and there needs to be some tests that need to be done. Could be a blood spot, 3-methyldopa, they may want a vanilactate to be done as well, but at some point it's very likely if they are suspecting a neurotransmitter disorder to consider a lumbar puncture. To consider taking the cerebral spinal fluid and analysing it for those HVA and 5HIAA markers of dopamine and serotonin.

It is important sometimes because there may be a clinical overlap in the way some children present. They may have AADC deficiency, but they can have another metabolic deficiency in that pathway, such as tyrosine hydroxylase or a terrin disorder I mentioned briefly, and they can also cause dopamine and serotonin deficiency.

So sometimes the doctors ask for a CSF profile to be requested so they can ascertain the level of the metabolic block, where is the deficiency occurring in this child they suspect of having a neurotransmitter disorder effecting dopamine and serotonin. Some of your children may have had a lumbar puncture and different labs across the world do it differently but it's really important that the clinicians work with the laboratories to get the diagnosis correct. In our lab we ask for 3 tubes to be filled in a particular order because if you go from the top of the brain here all the way down, there are different levels all the way throughout the spinal fluid in the spinal column. You must use the same fraction of CSF and relate that to unaffected reference ranges as we call them.

Tube 1, the first half mil that is taken from the patient by the lumbar puncture is to measure the dopamine HVA metabolite, the serotonin metabolite 5HIAA, so we remember those hopefully from the pathway.

The second half mil we measure the 5-methyltetrahdrofolate, you know that's important because it can be deficient, and the pyridoxal phosphate the vitamin B6 active form that's also required for AADC deficiency.

In the third tube we measure the terrins just in case we are looking for a terrin disorder.

And we have some very clear instructions for the doctors. These molecules breakdown very easily and so the samples are dropped into liquid nitrogen and frozen immediately at the bedside.

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This is something I always talk about from a laboratory point of view, it's about following the instructions and talking about communication from a neurotransmitter point of view, but the communication between families, clinicians and laboratories is absolutely essential if we are going to get this right and what we want to do is get this right first time. You really don't want to have to do a repeat lumbar puncture when it's not necessary because I'm sure for those of you who have children who have gone through this it is a stressful process.

Slide 11 Here we can play spot the difference. This is what we call a chromatogram.

Basically we are putting a patients spinal fluid, so the fluid from the lumbar puncture onto a system that can separate out the chemicals that are present in the patients sample.

So this is time along the bottom and going up is the amount of the particular chemical. We don't have to worry too much here, but here's a 5HIAA and a HVA, very nice, very clear, that's unaffected. I can spot that immediately and say that is a normal profile, Let's move down and now we can play spot the difference because where the 5HIAA was in this AADCd patient it's almost absent and where the HVA, the dopamine metabolite, is it's very low indeed. But what we've got is something new here that's building up. So we've got the deficiency, the down arrow, the HVA and the 5HIAA but we've got too much of something else and that is the 3-methyldopa coming from the L-Dopa building up because there's an enzymatic block. You can see it there but it's absent there. And this other one 5-hydoxytrytophan building up again because the serotonin pathway is impaired, it's got nowhere to go so it starts building up.

You can see in the laboratory AADCd by looking at what we call these chromatograms, this separation of chemicals and seeing the deficiencies and build up, very quickly we can say this is AADCd. Or we have a strong index of suspicion that it is AADCd.

Slide 12 Some of you may have heard of prolactin, it's another one to think about. It's not a perfect marker but it can be a helpful marker. Dopamine has many roles within the central nervous system and one of the things that dopamine can do is it can suppress the amount of prolactin that is produced. Dopamine is a brake, it puts the brakes on the production of prolactin, so if you are dopamine deficient, Page 11

there's less dopamine, less inhibition, the brake is no longer on so much and therefore more prolactin is being produced. So it can be helpful but we have to be mindful that there's many things that can conspire to alter and elevate your prolactin levels.

But when you are putting everything together and you've got a patient with a very strong clinical picture, you've got 3-methyldopa elevated, some valinactate, you've got the AADC enzyme perhaps measured as well, you've got the prolactin that's elevated it all helps you put everything together. Sometimes we request the prolactin to see whether it's elevated or not, because it can be informative but it's not a perfect test but it's one that we add on or we ask to be considered when we are building up the case. But the message here is less dopamine, less inhibition of prolactin production and therefore you produce too much prolactin.

Slide 13 Now I can show you a real result because hopefully my training programme has worked and you understand the highers and lowers so I can give you a genuine case and we can talk through from a point of view of the laboratory in how it happens. This is a real case of a five month old.

What I am particularly pleased with in the UK, and hopefully around the world, is how much quicker referrals/diagnoses are made. Five months is much quicker than it used to be. A five month old child was referred to us because of what they told us over the phone with regards to their results. So this is a lab quite a way from us in London, rang us up because they were doing a urine organic acid profile and said that the patient had a very high vanilactate. Remember that's on that pathway that goes from 3-methyldopa to vanilactate. So they were suspicious. So we said this looks very interesting do you have any clinical details?

So they rang us and said the patient has developmental delay, a movement disorder and something strange is going on with their eyes.

I told them you need to really think about AADC deficiency and we suggested they go very quickly to a lumbar puncture and very quickly indeed we got the samples, turned it round and these are the results we got.

Hopefully this all makes sense from the metabolic pathways that we looked at. In red are the results which are outside the reference range. You can see HVA was virtually undetectable, on the right hand side of that value is what you should typically see in an unaffected individual, the normal range, the reference range.

So HVA very low. 5HIAA also very low because it's coming from serotonin. Look at the 3-methyldopa, it's very high because it's before the enzymes, so it's high and it's building up and it's going on to make vanilactate after all, which is also high Page 12

in the urine.

So the biochemistry that I have shown you is hopefully manifesting itself in it's true result. You can see that hopefully what I've been telling you is true. The 5-methyltetrahydrofolate in this patient was not decreased. That's important because it's not always decreased. We've got patients where it's low and patients where it's normal. It's something that needs to be thought about and that's something that the clinicians are aware of whether to or whether not to treat with folinic acid which is the right form to give to replace any deficiency of 5-methyltetrahydrofolate. It may be that for this individual they need to monitor it because look at that high 3-methyldopa, 2023 there, it may be that if that pathway is continually making all this 3-methyldopa then eventually you could run into some problems with you folate availability, because you need that 5 MTHF to make the 3-methyldopa.

Some patients may be lower at the first LP, it may always be normal. It is a difficult one for us to be absolutely definitive upon. The patient in this case had elevated prolactin, we know it's dopamine deficient therefore the prolactin is elevated. We measured the enzyme activity. We can get our patients plasma and we can see how quickly L-Dopa is converted into dopamine. It's a rate, how fast does it go. It should go reasonably fast in a controlled unaffected individual. In a patient with AADC it would be almost non-existent because the enzyme is not there. And then you do the genetics to confirm the diagnosis and obviously inform the family of the mutation that's causing the deficiency.

So that's it. That's the biochemistry of AADC. Hopefully I have demystified the biochemical pathway but also the mindset we go through in the laboratory and how we put our knowledge of the biochemistry into action to make that diagnosis of AADC deficiency.

Q&A Julie Do the biochemistry results tell us how severe the disease will be?

Professor Simon Heales The short answer is not really, no. That's the problem is here. We are looking at a snapshot when we do the lumbar puncture and look at the HVA and 5HIAA. So that's the sort of levels we see at the time the lumbar puncture was taken. So it can give us the diagnosis but I don't think we are at the stage where we've got a Page 13

correlation between the biochemistry that we are seeing and the potential severity of the clinical outcome. I think that we are not there and it may be a long time before we are there and it may come more from looking at the genetics and the biochemistry together. But no you can't really. We never say this is a very mild form or severe form on our reports. We just say this is very indicative of AADCd.

Julie Does folate deficiency only show up later in life or can it be deficient from birth in AADCd? Professor Simon Heales What a great question. I think I alluded to it a bit. It's frustratingly irritating, no disrespect to folate, but in some patients, at the first diagnosis they have had a low methyltetrahydrofolate, others it's been absolutely normal, as I showed you in the 5 month old patient. So I think it's something we need to be made aware of because it's something that can happen. It's coming because you are making so much 3-methyldopa and most clinicians, I think the guidelines recommend that you do consider folinic acid because it has the potential to be deficient and I think it's something that the clinician needs to talk with the families about giving it because there will always be that the potential that the folate can be depleted. But it's a bit of a variable beast. It doesn't happen all the time. It may happen in that five month old and if they hadn't been given folinic acid later, on it could cause problems, so I think it is something that has to be monitored. It's a bit of a risk then, it's a balance. You've got to always consider what is best. Do you give folinic acid and therefore prevent a potential deficiency or at some point do a lumbar puncture and get the evidence to definitely give folinic acid. I think that's a conversation that you need with your clinicians. There is good chemical reasoning to keep an eye on the folate status.

Julie Do folate rich foods help?

Professor Simon Heales I'd like to say categorically yes but the problem we've got here is where the 3methyldopa is produced. If a huge amount in the brain we need to replete it and it's got to be the right form. So yes I think all of us should have a goof folate diet anyway, however I think it is something that we, it would be wrong to say that it didn't help, but I think what we've got to do is carefully keep an eye on this and I think you may need a supplement as well as having a healthy diet. I think in some Page 14

of those patients the levels could get quite low and so you need a supplement as well. I think it's something you need to consider with the clinician. Most of them are aware about this pathway and that it needs to be kept an eye on folinic acid.

Lisa Are normal HVA and 5HIAA equally balanced to each other, so the 5HIAA is 50% less than the HVA, is it always equal?

Professor Simon Heales Everything in life is very much in balance so, beautifully in balance. Those of you who Goldilocks and the three bears, it's the porridge - either too hot, too cold or just right - it's all about being just right. It's all about being just right and that's how our bodies work homeostasis getting everything into balance. 5HIAA / HVA ratio are reasonably tight they are between 1 and 4 in the CSF. So that's the range that we operate under normal condition. So 1-4 is that ratio between those two molecules so they can be quite equal or there can be slightly more of one than the other. They do tend to work quite closely together and correlate each other as well.

Lisa With gene therapy we know that the HVA increases but the 5HIAA stays low, does the difference between these 2 metabolites cause problems?

Professor Simon Heales For the other neurotransmitter disorders we have always been blessed by the fact that you can give a child the L-Dopa like in tetrahydroxylase deficiency and they get up and walk near normally. That's a slight exaggeration but it's life transforming. AADC has always been this challenge to get a good treatment, so gene therapy is a fabulous way of opening up now. I think it's early days Lisa because it's targeting the dopamine system, so you are correcting the dopamine pathway and that is something that should be celebrated. But, you saw on my slide, the consequences of the serotonin deficiency as well and so from a biochemists point of view it seems that the dopamine pathway is being corrected but at some point, as we refine treatments, we need to keep an eye on serotonin as well. You and I both know, we have to remind some people, not the people in this room, that there are other neurotransmitters apart from dopamine. The gene is called the dopamine decarboxylase gene and people forget about the serotonin pathway. I think it is something that needs to be addressed. I am so Page 15

pleased to see a treatment coming through now, ok it's looking at the dopamine pathway, but as we progress this treatment it has to look at the serotonin pathway as well because they are working together those neurotransmitters.

Lisa Does the increase in dopamine after gene therapy also increase norepinephrine and epinephrine?

Professor Simon Heales I haven't seen any data from that because I've only looked in the CSF and we only measure the HVA coming from the dopamine. In theory you should see the other neurotransmitters being produced as well and I think that's an incredibly important question and we need to look. Those countries that have looked into the CSF, because there are metabolites of norepinephrine and epinephrine that you can look at in the CSF and perhaps we need to have a chat with all the labs to see if they are seeing that as well. That's a really good question.

Lisa If it doesn't increase further down, norepinephrine and epinephrine, you do leave the autonomic system dysfunctional so that would be really good for us to have.

Professor Simon Heales It's a superb question. I have written it down as an action point. We do focus on the dopamine and I think what we've managed to do as a Trust is open up people's minds to look at other neurotransmitters as well and I think it's a really good question Lisa.

Lisa Does the measure of peripheral metabolites equal the measure of brain metabolites?

Professor Simon Heales We have useful biomarkers so the 3-methyldopa does appear to be proving it's worth, so that's something in the periphery that is telling us that as a screen that the patient does have AADCd. That's a good one. The vanilactate in the urine, is also one that could be useful that you could look at, always with a degree of caution when you are outside the central nervous system because things aren't Page 16

exactly the same but 3-methyldopa is looking quite useful to identify the patients.

There are other things that don't quite correlate. In some AADC deficiencies there is a paradoxical finding in urine where there is actually an increase in dopamine production. It's called a hyper dopaminurea which means there's lots of dopamine in the urine it looks like there is a peripheral metabolism of L-Dopa that is building up and it is giving dopamine in the urine which is the complete opposite and counterintuitive to the biochemical pathway. So it’s at variance to what we know is happening so we have to tread cautiously when we are outside the central nervous system but also exploit those markers that are of use. And of course you can measure the enzyme in blood as well. In the central nervous system you can see the degree of deficiency that you've got in the brain and of course post gene therapy you want to know that the brain has been corrected.

There's a use there.

Lisa That relates to folate as well, measuring folate in the blood as well can be normal?

Professor Simon Heales Absolutely. It's also the type of folate. There is a family of folates. They are all slightly different folates and they are all measured as a group in the blood usually by hospitals, whereas in the brain we are measuring one folate, what we call a methyl donor, it's giving up its methyl to L-Dopa to make 3-methyldopa and that's the one particular folate that we measure. So again you have to tread cautiously about peripheral folate verses the CNS folate .

Lisa I think that is because many of us are with doctors who, if they get a normal level or even a low normal level, wouldn't consider that could still be deficient in the brain, so that's always the complication.

Professor Simon Heales And the other one is to make sure they think about folinic acid and not folic acid.

Folic acid is artificial and it can make the CNS folate even more deficient so it's folinic acid and most doctors do know about that.

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Lisa How often do we measure whole blood serotonin to know whether that is deficient equal to the brain deficiency?

Professor Simon Heales Very rarely nowadays. We used to measure it quite often back in the day with Keith Hyland and myself, we measured serotonin but now we don't measure that.

It's something I think we should do but I think it's something, we need to open up and really start championing serotonin. We don't do it that often. What we tend to do in the blood is the enzyme and confirm the diagnosis. We are not doing enough justice to serotonin at the moment.

Julie Would you recommend to check the overall methylation cycle for AADCd kids?

Our son has very low amino acids in liquor and cystine is high.

Professor Simon Heales It's how you interpret those results, that's the question here. You can get a whole raft of results back and because we are still learning, if you like, about AADCd, I think the methylation cycle is clearly important, that's where 5-methyltetrahydrofolate sits. It has a pivotal role and if you become deficient in that you are going to impair various methylation cycle steps. The thing is where do you look for this and I think it would have to be in the CNS, because that's where most of these reactions are occurring and we don't have a lot of methods at the moment to look for the methylation cycle within CSF. It's something that we are trying to set up. There are all these molecules s-adenosyl methionine and s-adonesyl homocysteine which form the methylation cycle. I just think that if you get results in the periphery on the plasma amino acid it's how you interpret and how you move forward with those results. Sometimes you get a lot of results and there can be some changes but it's what do you do with those and I think that's why you need to make sure you are working with a centre that has laboratories that understand this condition and clinicians that also understand it because results are only useful if we interpret them together. These are not easy, none of this is easy, you have to do it together and so I just think when we look in the periphery just be careful it doesn't reflect. But the methylation cycle could arguably be important to review because what we are talking about is 5-methyltetrahydrofolate being passed off the methylation cycle. Very good question.

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Julie We have been told that we could try taking 5HTP supplement where could this be helpful in regard to what is shown on your charts?

Professor Simon Heales 5-hydroxytrytophan is the precursor so you've made it but you can't go and make serotonin, you've got the metabolic block. So to give 5-hydroxytryptophan to most of our patients it would not have the ability to move to serotonin. So you'd be building up more of something that you don't want. It's like L-Dopa. I said tyrosine hydroxylase can be treated with L-Dopa but frustratingly most of our AADC patients can't be because of where the metabolic block is. So 5-hydroxytryptophan frustratingly is going to hit a wall, it's not going to go through the AADC enzyme. So therefore from a biochemists point of view, I would struggle a bit for most AADC patients to be given 5-hydroxytrytophan.

Lisa Can I add to that because yesterday we learnt from Mita there are some mutations that could respond to L-Dopa. Those that could respond to L-Dopa could they potentially respond to the 5HTP as well?

Professor Simon Heales Very good question. That's why I said most of our AADC deficient patients. What I think is happening now is we are moving excitingly into personalised medicine here because you could grow a patients cells and feed them L-Dopa and see if they are one of those rare of the rare patients that has got a mutation that allows them to convert L-Dopa to dopamine, not a well as under normal conditions, but it's still a possibility and you do that by showing that is the case.

You would then have to do the same by giving 5HTP and seeing if it gets converted into serotonin. I think you need the evidence for it before you just go ahead and give it. I think that's probably what you were talking about yesterday, is there is a rare form of a rare disease where you may just be lucky enough to convert the L-Dopa to dopamine and then you need to demonstrate that the 5HTP can be converted to serotonin. But my biochemical advice would be don't just go ahead and give 5HTP without knowing a little bit more about whether you can convert it or not. You have to be very careful.

Lisa Some of our families that have had gene therapy are showing higher levels of Page 19

HVA. They no longer take dopamine agonists so is it possible they could be treated with SSRIs - Selective Serotonin Reuptake Inhibitors and would this increase 5HIAA? If they are having lumbar punctures it could be promising to look at including an SSRI and whether that result could be looked at the same time that you are looking at the HVA.

Professor Simon Heales Absolutely. In theory yes. In the case that I showed you that patient had undetectable 5HIAA, the serotonin levels were extremely low to start with. In other cases you may have a slightly higher level, then you are preserving any serotonin that's being released by using these various pharmacological agents.

So I think it's possible but it needs to be done very carefully with a clinical interpretation as well. I think what's obvious Lisa and Julie and others is that we are beginning to talk a lot more about the serotonin pathway and we have got to start addressing that as well.

Julie How does an anticholinergic like Benztropine help AADC?

Professor Simon Heales I don't know how that is going to work exactly. There is interplay with the neurotransmitters as you'd expect. We know there is interplay between dopamine and serotonin. We know there is interplay with the acetylcholine system as well.

With Parkinson's disease if you target the acetylcholine pathway that has a beneficial effect. We know that in Parkinson's disease we are dealing with a deficiency of dopamine so there is some rationale behind that but it's a little bit outside my comfort zone to say whether that would fully work or not but I can understand the rationale for Parkinson's and I can understand the rationale that neurotransmitter pathways do interact and support. Again you need the right communication with the right people.

Julie Bromocriptine lowers prolactin level and prolactin raises dopamine, can you explain this cycle?

Professor Simon Heales It's a good question. When you don't have enough dopamine, you don't have dopamine binding to receptors, so there is less receptor activation and therefore Page 20

prolactin levels go up. Bromocriptine is a dopamine mimic. It's not dopamine itself but it tricks the body and it binds to the receptors. So it can actually bind to the dopamine receptors and mimic the action of dopamine. Because it's mimicking the action of dopamine, it can do what dopamine does in this context and therefore the prolactin levels start to come down again. Dopamine suppresses dopamine production.

Bromocriptine acts as a dopamine agonist if you like, it's like a friend of dopamine it can bind to its receptor. When it binds to that receptor it does all the jobs dopamine can do and that will bring the prolactin down. So when a patient is on bromocriptine their dopamine levels will drop because it's a dopamine mimetic, so it's mimicking the action of dopamine, so that's why you will see the prolactin come down. You've got something that is working like dopamine. Bromocriptine is dopamine like.

Julie Is there any harm in giving too much folinic acid i.e. Leucovorin?

Professor Simon Heales Again I haven't seen cases of overdoses of folinic acid. We have very clear dosage recommendations. I never give out advice on how much you can give my comments would say consider treatment with folinic acid. Again it is about having everything right in the right window of normality or what our reference range should be. I can't remember seeing many, if any, AADC patients who have been on folinic acid where the levels have been too high. They have always been comfortably within the reference range when they are on treatment. Again I think that's a really important conversation with your clinicians. They may want to titrate in the folinic acid and not rush it too much, they may want to up the dose as well. If there is a clinical response, and that's usually from the mum or dad who sees this, they say yes the folinic acid is working, that's really important. You might not need to go for a lumbar puncture because you know it's having a clinical effect and I don't know if people have seen benefits and clinical effects from folinic acid. You have haven't you Lisa, that doesn't mean we need to do another lumbar puncture on Jake it just means we need to keep his folinic acid levels, we need to keep giving it. The other this to know about these neurotransmitter disorder treatments, is that they are given on a weight basis, a kilogram per kilogram. So if the child goes through a growth spurt, puts on a lot of weight etc then you need to monitor the dose to make sure you are giving the same amount per kilogram. Sometimes you do get some slippage in some of these treatments because the child has gone through a growth spurt. All of this is conversation with your doctor. If there's a nice response going on with the folinic acid then that to Page 21

me that makes a lot of sense and you don't need to do a lumbar puncture but some people want to have a lumbar puncture first to see if it's evidence based. I think it is a decision between families and their doctors.

Julie Another one on 5HTP. Would 5HTP be helpful after gene therapy?

Professor Simon Heales That's a fabulous question. They're all fabulous. I don't have a favourite. It's a good question because you're putting back in the enzyme, you are putting back in AADC, fantastic. And therefore we are seeing the increase in HVA. The important thing is the brain regions it is currently being put into. It is being put into dopamine rich areas. So you are correcting the dopamine deficiency. What we've got to be very careful from a biochemist point of view is that we don't end up creating new neurotransmitter regions in the brain that are not normal. So if we were to give 5HTP to where the enzyme is being corrected and is dopaminergic rich, we could also make that serotonin rich as well. But that might not be the appropriate compartment so I think we've got to be careful how we tread. Biochemically if you gave 5HTP to a patient that had been given gene therapy you would probably make serotonin as well, however you've just got to be careful that you could be making it in the wrong region of the brain and that can cause all sorts of problems. So whilst it's tempting, from a biochemists point of view, I think you have to step back and say actually what we need to do is start targeting other regions of the brain and then we can start seeing the corrections of serotonin. So I know it's something that I know clinicians and scientists around the world are very aware now that we have got to address the serotonin system as well.

Lisa The 5HTP and the SSRIs would act differently wouldn't they. The SSRI would recycle what exists already whereas the 5HTP is pushing to produce more natural serotonin.

Professor Simon Heales Absolutely, you'd be putting in the precursor, you've opened up the block, but you may have opened it in an incorrect region. If you were to give a SSRI that would be more global across the brain and you'd naturally enhance the serotonin in the areas where it is deficient. It's getting it in the right place at the right time. I'm overwhelmed in what I'm seeing. We've moved into an era where we are making Page 22

the correction of one neurotransmitter but of course it is mark one of a product that is going to have to change and get better to address the serotonin pathway as well.

Julie Does the body have alternative ways to produce dopamine and serotonin?

Professor Simon Heales They are all good biochemical questions. With regards to the dopamine pathway there are some alternative pathways that have been described but perhaps not so predominantly in the central nervous system so. If they were you'd perhaps get a degree of correction of the defect, we may have a pathway that could compensate, but we are not getting that compensation. That doesn't mean to say we can't exploit those other pathways, enzyme replacement therapy or look at different ways of enhancing pathways. I think that's what we always do. We always look to see if there are other ways to boost the pathway, but I don't think we are blessed with enough alternative pathway capacity at the moment, or that we can exploit to boost the dopamine and serotonin metabolism. Otherwise, obviously if there were alternative backup pathways, we wouldn't have such a problem as we are dealing with. So in theory there are some alternative pathways but they don't seem to be sufficient enough to correct the defect.

Julie Can PDE4 inhibition, Phosphodiesterase inhibition improve AADC enzyme activity?

Professor Simon Heales Not to my knowledge, but my knowledge is limited. I have never seen anything around that at all. I mean of course we are always trying to boost residual enzyme activity. That's something are always keen to do. If we understand how the enzyme works, in the absence of a gene therapy, let's try to boost what we've got. If there's a little bit of enzyme there, that's better than nothing, so let's try to boost it a little bit more. So anything that increases the activity of what we've got could be something that could help our patients. So that's why we think about pyridoxal phosphate, it's the essential partner for the AADC so by giving a bit more pyridoxal phosphate, perhaps you can boost what enzyme activity you've already got. So I'm not answering the question on PDE4 but what I'm saying is, it's a pertinent point let's try and activate what enzyme we've got and currently are doing that with B6 and I don't know about the PDE4 in addition. But anything that can target, improve and boost so that you've got residual enzyme activity is going to hopefully Page 23

give a better trickle through, because it probably will just trickle through to the dopamine and serotonin. And as Lisa said then think about giving SSRIs to try to boost what you've already got or MAOIs to stop the break down of what you've got.

Julie For Parkinson's dyskinesias they use CBD oil and Glutathione and say it works.

Would you be so kind to share your thoughts on it for AADC kids?

Professor Simon Heales It's about the nature of these two disorders. Parkinson's is a neuro-degenerative disorder so unfortunately patients with Parkinson's disease their neuronal cells are dying away and therefore you want to stop the process of cell death, neurodegeneration. Now it's all about the cause and therefore the best treatment. I don't know about CBD oil. People are making lots of claims about it and I have no idea at all. Glutathione however, is an antioxidant and in Parkinson's diseased brains we know they have a deficiency of glutathione, so you are correcting the defect. In Parkinson's disease, it's a multifactorial disease, that is causing it but one of the things they think is very important is oxidated stress. This is producing horrible reactive molecules that can cause havoc within the cell. To stop that happening we give an antioxidant to nullify the effect of these damaging molecules. That is part of the disease process and you are correcting a known deficiency. They are deficient in glutathione and you're correcting it.

So whilst there is overlap in Parkinson's in the clinical manifestation sometimes, and they both have dopamine deficiency, in AADCd it's a primary genetic disorder, not really associated with a neuro-degenerative process. That's a good thing because we want to be able to correct the defect, the neuronal cells are not dying. So I think it's all about evidence based, I think that if there is a degree of evidence that glutathione is deficient in Parkinson's then that's worth correcting.

To my knowledge I haven't seen anything around AADC and glutathione deficiency and therefore the evidence base for me isn't so strong for using that treatment. Different disease processes requiring different interventions. I'm afraid with CBD oil I just think we need to be very careful that we don't get too carried away with the hype. I work with other disorders as well and this constantly comes up. I'm a big fan where we possibly can of using evidence based and of course my evidence based comes from the biochemical pathway but I'm not taking away from the families who can see a clear benefit. I am only looking at the Page 24

biochemistry and importantly here is the families and what responses they are seeing. But the glutathione story is different in Parkinson's to what it is in AADC.

Julie Does using Zinc and Vitamin C make other drugs we use more effective?

Professor Simon Heales I don't know for AADCd. There's evidence for other drugs that you can take Vitamin C to help with preserving some drugs. I don't know for the AADC story and either for Zinc at all.

Lisa Yesterday we learnt that some of the mutations are unresponsive to B6, so given some of our early research that we did, is it still beneficial for them to take B6 if you are considered to be unresponsive to it?

Professor Simon Heales I think it's how you define unresponsive. We know that in unaffected cells if you give B6 you can increase the production of AADC enzyme. We are again going back to this personalised approach where we need to look at a patients cells, give them B6 and see if there is any improvement to their AADC deficiency. We are still treating a bit blind aren't we. We are hopeful that if you give the B6 it's going to work for the patient but in the end we are going to have to move forward.

We will get there Lisa and everyone because we will have a data base of mutations and we will know which mutations will respond well to B6 , which ones will be more at risk to the folate deficiency, we will get there in the end but at the moment we are hoping to increase any residual enzyme activity. At the moment we don't know who is a responder and who is not a responder and so I can see the temptation to give B6 but I suppose it comes back again to the clinical response. If there's a marked response in the patients and the families feel it’s beneficial, then it might be beneficial and worth carrying on. If it's not beneficial then perhaps it's worth not carrying on. I think at the moment we are relying on the evidence coming back from the families and the patients but in the future I am very optimistic that we will be doing testing on the patients cells and looking at the mutation database to say this is one that may respond to pyridoxal phosphate.

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Lisa With induced pluripotent stem cells are we moving to a point where we can avoid lumbar punctures and actually create personalised induced pluripotent stem cells from skin fibroblasts or blood and that we can try medications on those samples rather than repeated lumbar punctures?

Professor Simon Heales Induced pluripotent stem cells can take months to grow up, literally months. One of the proudest things we have done as a Trust Lisa and Julie is that we have raised awareness, from a situation where, you know from your own stories, it can take someone years to get a lumbar puncture for AADC, we are now talking about kids now less than five months of getting a lumbar puncture and getting a diagnosis within a week of a sample being sent to us. So we've got that ability to do it very quickly now to get diagnosis and get the available treatments in place as quickly as possible.

Currently I think because of the time it takes to grow up the induced pluripotent stem cells, to get them to dopaminergic cells, we are talking quite a long time.

You'd have a ticking clock where you didn't know what the diagnosis was. The reason a lumbar puncture is central at the moment is because clinically the patient looks like AADC but it can also tetrahydrobiopterin deficiency and the treatments are very different. But from a clinicians point of view they can't tell the difference.

Genetics will get faster and faster and you'll be able to diagnose much quicker, with that to get a definitive diagnosis but eventually what you will need to know, and we do have this sort of paradox in a lot of metabolic disorders, you can have the same mutation but one patient is much more effected than the other. It doesn't make sense, it should all be the same but of course none of us are the same and then the level of your dopamine and serotonin deficiency may be different even though you've got the same mutation as somebody else who doesn't need much correction.

There will always be a place for the lumbar puncture as we are trying to refine our treatments and check there has been a correction, but we can use other tools as well to perhaps make sure we only do it when it is absolutely needed. I think pluripotent stem cells from my point of view will be really important for understanding disease mechanism, trying out new treatments. But if you have a patients cells you can then feed them B6 and if it boosts you can say actually there is evidence that you need to give B6 to this patient or this patient is using a lot more folate than we thought so therefore you need to think about folinic acid.

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I think we may get to that situation.

Lisa We know that quite a lot of families would be willing to donate samples for research and we know that it takes sixty six days to nurture an induced pluripotent stem cell but can you store them once they've matured? I know it's complicated, you can come back to them and they've all died, but if you can get to that maturity can they then be stored?

Professor Simon Heales I don't know. Before you've converted them, I think yes they can. I don't know enough about once you've differentiated them can you then store. I will ask Manju that because then you can have a bank of cells and store them up. I think we always have to cull our enthusiasm a little bit with these things. Remember what we are doing here, we are making a group of dopaminergic cells, and these induced pluripotent cells may have come from skin fibroblasts originally, what you've now created is dopamine making cell.

Remember in the brain it's not just a plate of dopamine creating cells we've got what we call glial cells around our neuronal cells, all these other types of cells that are working with the neuronal cells so we have to be careful that we don't lose sight of what's really happening in the brain. There's so much communication going on, with different cell types, the serotonin pathway, I talked about acetylcholine earlier. All these interactions may or may not occur when you are in the cultured situation. It's wonderful that we can take someone's skin cells and make a dopaminergic cell but we haven't made a mini brain so I always caveat with a degree of caution to say it's incredibly exciting but let's be careful we don't get overexcited.

Lisa I guess that when we have other markers as well, like the lumbar puncture markers, the genetics, if we create induced pluripotent stem cells and we bank that, you have as best a picture as you can get a swell as clinical presentation for each of those children, so if you have some back up information that supports that, that to me seems like a natural development. To see if we can store developed induced pluripotent stem cells to work on, along with the other data, that then we could maybe further enhance knowledge.

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Professor Simon Heales I completely agree and I think all knowledge is power isn't it. What we are talking about here, which is very much what we need total about is, personalised medicine. Not seeing every AADC patient as exactly the same. They have the same biochemical problem but that is a variable problem. Each patient is individual and when we move to this when you can use those cells to ask questions for that particular patient, is something I think we are going to see. We are going to see, as we look forward, that there will be much more emphasis on personalised medicine and the genetic sector, etc so we can actually tailor treatment appropriately. It's very exciting.

Lisa Personalised medicine is going to be very good for those that are severe or those that are not really good candidates for gene therapy. Would the gene therapy alter the state of an induced pluripotent stem cell if you took a sample after?

Professor Simon Heales It depends where you get the sample from. We've done a lot with fibroblasts haven't we. Obviously gene therapy is going to the brain and therefore you're taking from the skin so you don't get the gene expressed outside of the region where the gene is injected so it would still be deficient outside of the periphery so that would be my prediction.

Lisa The step before pluripotent stem cells, somebody has asked that they have frozen stem cells from their son that they had collected from placenta at the time of birth, frozen in a stem cell bank. Is that useful for research because they could donate it?

Professor Simon Heales That's such a nice comment and I think everyone on this forum recognises the power of research to help us sort this problem out. I think that's something that should be shared with some of the laboratories Lisa that are working actively on the disease and something. I think is incredibly valuable and a generous offer.

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Lisa That's not the first time we've had that offer but we've never known where to bank it and who's going to make good use of it so if we had somewhere where that could happen.

Professor Simon Heales Perhaps that's something we should talk about at the next Trustee meeting. I think if you are getting offers like this we really want to use those appropriately. It's a very nice offer and we need to look at seriously to make sure we use it appropriately.

Lisa Thank you Professor Simon Heales for joining us today and helping us understand more about our children's biochemistry. It's been insightful and we are very grateful to you for that.

Also thank you to everyone here for joining our fantastic professionals workshops today and all week. Our experts have done us proud and we hope they have provided you with useful information and tools for all our families to manage their own children's conditions in a more informed way.

A very special thank you to you Julie, who has worked very hard to create and celebrate this awareness week, and our 15th birthday by designing all our children's profile pictures, encouraging ways to donate, and you've even built a special video that will be released soon as well so we look forward to that, highlighting the Trust's journey over the past 15 years.

Thank you to everyone who has worn BLUE this week raising awareness and much needed funds and that just enables us to champion on behalf on your children behind the scenes.

A big big thank you. I hope that everybody has a good morning or afternoon or evening wherever you are in the world and we will feed these back into the group and we can develop these conversations at another time.

Simon is there anything else that you want to add before we close.

Professor Simon Heales It's a real privilege to work with you and the families so thank you very much.

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AADCd Family Workshop 5 : Transcript Produced by Julie Ramsay Verified by Lisa Flint & Professor Simon Heales Translations by

teamextended.com

The AADC Research Trust would like to thank all involved.

#CureAADC #CureAADCd Page 30