AADCd Family Workshop 4: Transcript Understanding Phenotype Vs Genotype in AADCd
Professor Mita Bertoldi Italy
Transcript ... Lisa Hi everyone, I know I have seen most of you over the last few days but I am just going to go through the opening again and welcome Professor Bertoldi to the session. Thank you all for joining today and thank you Professor Bertoldi for joining us. My name is Lisa Flint, founder of The AADC Research Trust and mum to 23 year old Jake who has AADC deficiency. 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. I am sorry, because of time constraints we can't introduce each and every one of you. The subject is Phenotype Vs Genotype in AADC deficiency is of extremely high interest and we hope you find this workshop helpful. Let's begin by welcoming and introducing our presenter. Mita Bertoldi is a professor of biochemistry and in understanding how proteins function and the relationship between their structure and function. Professor Bertoldi has been a friend and collaborator with the Trust for over ten years and works at the University of Verona, Italy in the laboratory of protein chemistry. I'm going to hand over to you now Mita for your presentation.
Dr Mita Bertoldi Thank you Lisa, for inviting me to talk this afternoon and thank you to everyone for listening to this lecture which could be a little technical but I tried to answer some questions that Lisa raised in the past few days and tried to point my presentation to what your interest is. I will try to go very slowly while speaking and please tell me if you miss something so I can repeat it. This was the title Lisa gave me; Phenotype Vs Genotype. I am really happy to be here to celebrate 15 years of AADC deficiency awareness.
Slide 1 The first question that Lisa raised was "what does it mean that AADCd is a single gene defect?". This concept will lead us to the concept of genotype and what genotype is. Page 1
As you know a gene is a stretch of DNA coding for a protein, for example the colour of the eyes is controlled by one gene, the colour of the hair is controlled by one gene. And as you probably know genes are organised on chromosomes inside the nucleus of each cell. Chromosomes are regions of very condensed DNA. Humans have twenty three copies of chromosomes inside the nuclei of all cell types. These chromosomes are divided into twenty two homologues and one sexual that determines the gender, feminine or masculine. This means that in our neuclei we have two identical copies of each chromosome. So genes are organised on chromosomes and in our cells we have two copies of each chromosome. Two copies of chromosome 1, two copies of chromosome 2 and so on. This is because one chromosome is of paternal origin and one chromosome is of maternal origin. So each gene is present on the maternal chromosome and the paternal chromosome. We have our DNA organised in chromosomes. The DNA is the depository of all information for the cells and we have chromosomes organisisng copies. So we have two copies of chromosome 1 and two copies of chromosome 2 and so we have a double copy of the gene. This is the meaning. Each chromosome, in a particular phase of cell cycle, is organising two sister chromotids, because they are dividing. This is not important.
Slide 2 The genotype are all the genes in an individual, all the genes present in a cell. The gene for AADC belongs, it is present on the short arm part of chromosome 7, and this is the entire genotype of a human being. This is a male because it has XY, a female will have XX chromosomes. AADC gene is present here. You can see each chromosome has two chromotids, two arms, because this is a dividing cell.
Slide 3 But in other cell types that are not dividing we have two copies of a single arm chromosome. It is not important if you see it doubled or singled. The fact is that on each one of these two, one of maternal origin, one of paternal origin, there is a little stretch. This is an enlarged scheme of this chromosome, the bands are colorometric bands, that tells us where exactly, like centimetres or meter, the gene is positioned. The gene for AADC is about in this region. Page 2
Each one has two copies of chromosome 7, one from the mother, one from the father, and each one of us has two copies of the gene located in this upper part of the chromosome.
Slide 4 In biology, from the gene, from the DNA, there is the transmission. The conversion of the message of the DNA into protein through two passages that are called transcription and translation. So the message that is present on DNA is translated into protein. The DNA, the gene, is made of nucleotides that are the single part, biomolecule, that form a DNA, and this message is converted into a protein that is made by amino acids. So the gene coding that is made for AADC, through normal biological process, will determine the formation of the AADC protein. It is the protein that exerts the function, in the case of AADC, for dopamine and serotonin. The phenotype means how the gene manifests. It is called technically, the gene for the colour of the eye can possess multiple alleles, because the eye can be brown, green or blue. The problem of the dominance and I tried to make a scheme of this. We call allele, or character, the manifestation of the gene message. It is the protein that makes our hair or eye colour blue, brown or green. I would like to show you an example. The genotype on the chromosome tells us that this is the position of the gene for the hair colour, but each person has two chromosomes, one from the mother and one from the father. In this example this guy has two chromosomes that share the same type of colour, phenotype. The gene is the colour of the hair. The phenotype is brown, brown here and brown here. This makes this guy have the brown hair phenotype. This ginger genotype makes this guy, since the two alleles that are the manifestation of the gene, hair colour, have the hair colour ginger. This is a more technical point of view. If you see the capital letter means dominant character, the lower-case letter means recessive character. This guy has two parents that have had the same dominant brown hair genotype. Each one has given one of it's equal gene, making the son identical as the parents. The same is true for this other. The two parents of this ginger haired phenotype have the same colour of hair. Each one gives the same allele, because there is no other possible combination, and the son the is homozygous for this character. Homozygosis means that we have the same allele, the same gene. Page 3
Slide 5 The dominance has been studied by the classical Mendel Laws. What happens when a person, for example, with brown eyes crosses with a person with blue eyes? We know that brown eyes are dominant over blue eyes. We have this one, this is the mother, for example, can give only the capital B, and the father can give only the lower-case b. So the son is a heterozygous because the two characters, the two alleles are different but the phenotype, since this one is dominant over this one, is brown. So we see people with brown eyes but we do not know if these people are dominant homozygous, or heterozygous. If two heterozygous cross with each other we have the possibility of having, for example, for the mother, these two types of inheritancy of gametes, or the father these other two types, and so we have this combination; brown eyes homozygous, brown eyes heterozygous, blue eyes homozygous. As you can see the genotype could be different from the phenotype because the genotype looks at the combination of the two alleles from the father and the mother. In fact, your kids have genotype with two parts, we will see them, one coming from the father, one coming from the mother. The phenotype is different because of dominance. For AADC deficiency, for each mutation, we do not know AADC dominance effect so we cannot predict phenotype exactly. It is something we can predict, but not exactly. This is the effect of dominance. If the mother has blue eyes, she can only give the same type of gamete. If the father has brown eyes and he is heterozygous, he can give two types of gametes. We have some that are 50% heterozygous brown, 50% homozygous blue. I am telling you this in order to understand the genotype and phenotype. For genotype it is easy because it is sufficient to do genomic analysis to see which mutation it is. If a son is a compound heterozygous I will show you later in my talk, he has a mutation, one coming from the mother, one coming from the father. The phenotype, is how the effect of mutation appears and this is governed by the dominance effect that presently are not easily understandable in AADC deficiency. It is different for the colour of the eyes, this is really easy. But if we have a protein, very large, that can undergo a lot of mutations, the information about phenotype is more and more difficult to obtain.
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Slide 6 Let's look at the DNA segment that causes the AADC protein. This is that little piece, I would like to go back to a previous slide - here I am looking inside here, inside the gene coding for AADC to let you know how it is organised. Each one of us, ours sons, our children, possess two chromosomes 7, one from maternal origin and one from paternal origin. The AADC gene is made of these squares that are the so called exons, this means the coding region. The DNA sequence inside of them and is put together and forms the protein. The link regions are not called a region, they are the so called introns. We can also have some mutation in these regions, for example the Taiwanese mutation is a mutation that involves the splicing region, that is a region that is borderline between an intron and an exon. I will come back to that later. So this segment of DNA on the chromosome 7 cause for the AADC protein is made of coding regions that together give rise after translation to the AADC protein. So in AADC deficiency there are two alleles of the same gene, one of maternal origin and the other of paternal origin.
Slide 7 This is one of these representation of the gene of AADC. These are the references that tell us that maybe the number of affected people is higher than we think. AADC is probably not so ultra rare as we think. These are the type of mutations. These boxes group mutations. When you see an asterisk, it is a deletion. The entire protein has 480 amino acids. This is the gene but it codes for a protein that has 480 amino acids. When translation stops at amino acid number 7, it means the protein is really short and degraded by the cellular systems and it is not functioning. It is this genotype, that is arginine 7 deletion, on the right you can find the mutation on the DNA that gives rise to the protein modification. The deletion, if it is too early, gives rise to a protein that is unable to bind the co factor, the vitamin B6, or pyridoxal phosphate co factor, and is degraded by the cell. Here in yellow I have pointed the residue lies in the amino acid, that binds PLP. So every deletion, before this point, gives rise to a protein that is degraded and so the patient has no protein. That allele makes no protein. What you instead see is a substitution, glutamine for lysine, valine for leucine or Page 5
arginine for histidine. There is an amino acid substitution. An amino acid substitution gives rise to a mis-sense. A mis-sense means that an amino acid that makes a certain function in the wild-type, in a healthy protein, is substituted by another that can cause damage. So point mutation, that is the conversion of an amino acid to another, gives rise to a mis-sense mutation because the protein it exchanges it's sense, in the sense it could not be able to form dopamine and serotonin. Also a single amino acid could be important for the function of the protein. We have a frame-shift, we have added these two letters 'fs', this is a mutation that has arrived. Tyrosine 37 that has been exchanged to threonine and then there is an alteration that has determined a different meaning in the protein so the following amino acid are exchanged and then when you see the asterisk this means that the protein has arrived to 37th amino acid and goes on another 5 amino acids out of the sense, not presenting the normal protein. There is a deletion of the protein and then the protein stops. So this protein is longer, 37 plus 5 means 42 amino acids and is degraded. The patient has no AADC protein. The same is true here. Proline 43 than becomes leucine that becomes a frameshift. It is called frame-shift because there is an insertion of a deletion of a single DNA base that determines a shift in the reading of the nucleotide, that do not code for the appropriate amino acid. This is under the process of translation, because the DNA is organised in codons. This means three nucleotides, and for each three nucleotides there is the corresponding one specific amino acid. The is called the genetic code. The translation of the genetic code will show a nucleotide triplet into amino acid. If the nucleotide triplet is altered by a deletion or by an insertion, we have a frame-shift of the reading and so the amino acids are all different from the original functioning ones. We have mis-sense mutations when an amino acid is exchanged for another. We have frame-shift mutations when there is a mis-sense and then a frame-shift, some different other amino acid for example 21, and then it stops because when the apparatus of protein translation meets a codon, that is called the stop codon, this apparatus is disassembles and the translation of the protein does not go on. We can have other frame-shift, for example, isoleucine 33 is exchanged with asparagine, a three letter code for amino acid and then we have a frame-shift of 60 amino acids, we arrive at 493 larger than the original protein and then this Page 6
jumps and we have the end of the translation. This is repeated when you find a frame-shift. Another type of mutation are the splice-sites. The splice-site, the best one is the Taiwanese, we have a mutation in the non-coding region and for this reason you do not have an amino acid reference. This mutation means that when we are at the 571 nucleotide, 3 cytosines were exchanged by guanosine and this is a language of nucleotides, we have an alteration, a mutation of DNA. But this is really harmful because it gives rise to a protein of 238, about here, amino acids that cannot go on with binding of PLP. And so the Taiwanese genotype is really harmful. It is really severe because those patients have no AADC protein functioning. There are about 84/85, and now in the newly reported data, literature, I saw that this number is probably increased to about 90 mutations and this gives rise to about 120. I think the patients could be more, basing on the new variants recorded in the last few years it could be 140. We think the number could be higher and higher. In the gene of AADC we can have some casual and random mutation that could give rise to mis-sense, frame-shift, splice-site and non-sense. Non-sense are the deletion. The protein is too truncated. It cannot go on and bind the PLP. So each deletion before the PLP can bind gives rise to a species that is probably degraded because it is unable to bind PLP. The patients could be homozygosis or heterozygosis, because we can say that this is one copy of the gene, for example of maternal origin but each one of us has two copies of the gene. If we carry the same mutation we are homozygous, if we carry different mutations we are heterozygous. I will come back to that in a few slides to show you this situation of homozygous and heterozygous genotype and phenotype in AADCd.
Slide 8 This is from a review that shows the genotype. The genotype frequency in literature refers to combination of mutations. You can see there is a double. The genotype is this part, with a semi colon (;), means maternal;paternal, maternal;paternal. And you see this is the Taiwanese mutation that does not give rise to protein. This is a normal zygous protein variant that is due to identical mutations on the two alleles, the two genes, one of paternal and one of maternal origin. And this Page 7
gives out the frequency. The Taiwanese one is about fourth of all identified AADC deficient patients. And then we have the other phenotype and genotype as reported by this author two years ago. You can see we can have homozygosis or compound heterozygosis. This means we have one mutation of one allele and one mutation of the other allele. This is no protein and one point mutation. This is called hemizygosis because we have two different genes in the genotype but one of the two does not produce protein, so the only protein present is the other one. So we have homozygosis, hemizygosis and compound heterozygosis. This is an example, homozygosis and this is compound heterozygosis. The allele frequency means the frequence of the single one. If we see genotype, this is one genotype, composed by the maternal and paternal origin. If we see allele frequency we must double. These are two allele, one of the mother, one of the father. Gene means the physical position on the chromosome. Allele means the single one. To see the allele frequency of this I have to count this as one, this as two and go on to see if some other one has, this for example, this is three and this is four. This is a genotype. The combination of two identical. This other genotype is the combination of two different. This is another different, the combination of two different but if I go and count the allele I must count one, two, three, four, five so the allele frequency of the Taiwanese mutation is more and more. We must double in the single gene. So homozygous is someone that possesses two identical alleles.
Slide 9 Once we know the genetic basis, the genotype, the genotype is not enough to understand the disease outcome. Here I have taken two tables that come from a paper by Tessa Wassenberg, in 2017, that show guidelines for AADC deficiency, and some of the symptoms. And another table that says the symptoms are tabulated as common, less common or non-neurologic. I would like to point our attention to the recommendations. The phenotypic spectrum of AADCd is broad. It is broad because we have a lot of symptoms. And frequently different symptom. It can range from very severe to mild. Page 8
When we have a patient with autonomic symptoms without movement disorders AADCd should be considered. There is no clear correlation between genotype, the fact of possessing two mutations, biochemical and clinical phenotype correlation, except for the Taiwanese because this is easy. They make no protein. Although, with some phenotypes that have alteration in the L-Dopa binding site, and we have associated, these few patients, with L-Dopa responsiveness. We have to be careful now, as this is four years old. So the message is, genetics is very important because it led to the identification of the problem on the gene and on the protein, but it is not sufficient because we have to understand what has happened to the protein. What is the result of a mutation, because this could tell us what the problem on protein level and if this problem is connected to symptoms, and this is my work. I try to understand how proteins function and I try to correlate this malfunctioning to severity of symptoms.
Slide 10 Here I would like to show you the classic example of the Taiwanese mutation. The Taiwanese mutation, as I told you, goes through a single chain of 238 amino acid instead of 480. I have represented the single monomer protein, to show you that the connecting part, the magenta one, is about half of the protein. This green protein, is what is done by these patients, will never fold or acquire this form because it lacks the other part that completes the structure. For this region, we find this black spot, are the protein and this is the functioning protein. This is the Taiwanese protein. It is recovered as insoluble in the cell fractions, because probably it does not biochemical fold, it does not acquire a structure that is responsible for the function. It's like having something that is not structured. You can have something but if it is not structured it can't be functional.
Slide 11 So I would like to clarify what is the situation in healthy individuals, healthy carriers, the parents of our children and in affected patients. Healthy individuals have two chromosomes, one from the mother and one from the father, and both AADC genes, that is both alleles, produce the polypeptide chains, the protein of AADC. These genes are called native, this means healthy. They produce wild-type polypeptides. Wild-type means healthy polypeptides and we have 100% healthy protein. AADC for functioning is a homodimer. This means that one polypeptide chain Page 9
interacts with another polypeptide chain and the interaction is random. This means that these two could be both of maternal origin, both of paternal origin or could be one paternal and one maternal, but it is the same because all polypeptides are healthy. They are not modified by mutations, so the interaction of the so called monomers, that means the single polypeptide chain, is really random and when all polypeptides chains are healthy and are identical to each other the association gives rise to AADC protein homodimers. We talk about homodimers because the protein for functioning needs to be a homodimer. This is a fully functioning, you can see in yellow the Vitamin B6, the PLP is bound. When PLP is bound the protein is functioning. This the situation for healthy individuals.
Slide 12 Healthy carriers, here I have plotted the deletion is a red cross, one point mutation, or the second point mutation. When the carrier presents the healthy chromosome that makes only healthy AADC protein, in the deletion this does not make protein, because most of the deletion occurs before the PLP binding site. So this is not producing protein. We have a healthy carrier in hemizygosis which has only homodimer functioning protein, homodimeric functioning wild type protein, but in quantity that is half of the original quantity. So we only have functioning polypeptide chains that form homodimers and the quantity is half. This is a healthy carrier in hemizygosis, so called because only one chromosome is functioning. This is the situation that is different from a healthy carrier that is in heterozygosis because one chromosome is functioning, forming wild type healthy protein, the other has a mutation. So we have two types of polypeptide chains. Native, wild type, a healthy type of protein, and polypeptide chain carrying the mutation. Each chain randomly associates with each other forming dimers so we have a complex population in the healthy carrier. Wild type homodimers, protein that functions normally, the homodimers that carry only one mutation in both chains and the heterodimers that carry one mutation in one chain and no mutation on the other chain. One big problem is in heterozygosis. This is a situation of heterozygosis, because we have no mutation and one mutation. This is not the problem, because this is a situation where an individual does not make this protein, so this is easier to understand and treat. There is only a reduction of protein, so the possible Page 10
symptoms of the parent are related to the decreased amount of AADC protein that is fully function. Here instead we have different protein population, because this chain makes healthy chains. This chain makes mutated chains, and they can randomly associate to form this combination and the problem of quantity. Can I say the quantity 20%, 25%, 50%? I cannot say, because the problem is dominance in heterozygosis. In dominance, we are deeply studying the effect of dominance but at this moment we cannot say the quantity. We presume that since healthy carriers do not develop AADCd it may be that having wild type of protein, this is dominant over mutation, but we do not have results because an intensive study on healthy carrier in heterozygosis has not been performed until now.
Slide 13 Let's go to the patients. We have two situations. We have a situation of a deletion, or a splice, where no protein is done, if we have a deletion on a chromosome, no protein is formed; if you have the same deletion on both chromosomes, no protein is done, so the child has no AADC protein. Or we have the same mutation on both chromosomes and these are the so called homozygosis patients that have only one polypeptide chain that forms the entire AADC population. These are the homodimers, and the homodimers can be easily studied because if we understand where the defect is and what it means, it is easy to suggest, to try to find a solution. The situation in heterozygosis is more complex because we have a hemizygosis condition, where one allele does not produce protein and the other produces protein but this protein is mutated. We have only one polypeptide chain, that is the mutated one, and less amount of polypeptide chain. Or we have two different mutations coming from the mother and the father and so we have a very different combination that are theoretically 25% homodimer carrying mutation 1 and 25% of homodimer carrying mutation 2 and 50% of heterodimer carrying both mutations. So the situation in heterozygosis is more complex.
Slide 14 I was asked to answer can we predict disease outcome? Disease severity can be predicted on the basis of the enzyme function in models. I go to the biochemistry to answer this question. We can predict the functioning of the enzyme. If we want to predict what the problem is we have to understand Page 11
the function of the protein, that depends on the structure of the protein. A structure modification in one part of the protein can determine a precise functional effect. We all know this protein, AADC, from L-Dopa and 5-hydroxytrytophan forms dopamine and serotonin that are essential neurotransmitters. This is the protein. I have shown you some of the mutations that have been identified and we have studied. We have to collect structural data to understand how the protein is arranged. These are technical biochemical approaches, but the meaning is we have to know what in the structure has been altered. For example, we have found that in this mutation here, this very early and terminal part, really causes big damages on the active site. Whereas mutations here are less damaging. So we can predict, collecting structural data, and collecting catalytic parameter data, that is functioning data, it is the amount of dopamine and serotonin. We can put these two data's together and understand how and which catalytic parameters are altered. Is this a problem of the chemical reaction because some step in the conversion from L-Dopa to dopamine has been changed or is it a problem of binding, because here the L-Dopa and 5-hydroxytryptophan are bound, but for example a mutation here prevents dopa binding or it alters dopa binding. The fact that less dopamine and serotonin is formed can have multiple causes so we are looking for the possible causes. If we understand the chemistry of the reaction it's ok, but if the problem is the binding we can suggest to the clinician to try a therapy including dopa and 5-hydroxytrytophan because this can help the patient to bind the substrate. The problem of the protein is only a binding problem, not a functioning problem, not a chemical problem. If instead we have a chemical problem in the reaction, if the amino acid that is crucial for activity has changed, this is another point because we have an incompetent protein. It is not able to rescue it's activity so we have to supply the patient with healthy, other AADC protein well functioning. It is important to understand at which level the damage, the mutation has exerted it's effect. I mean which amino acid has changed, what is the substitution and what does the substitution do to the structure and the function of the molecule. And assign each mutation to a specific protein region. We have understood thatthis region is really connected by the studying of the pathological, the Page 12
pathogenic mutation, this region is really connected to this one in a sort of domino effect to the active site. So if we change something here the protein does not function, if we change something here the protein does not function. If we change something here, it's not so bad. The fact is it is not so severe. So we can find a regional to correlate severity of the mutation on the protein AADC. This does not mean we can correlate severity to the symptoms but we are on the pathway to do it, because when you understand how the protein is controlled we can think and counteract this effect. Our aim, and we are doing this, we are collecting the final data that is almost ready to be presented, that can correlate severity and mildness of the pathology to the severity and mildness of the structural modification that effects function of the protein. We are looking to find correlation, the link between symptoms and protein structure and function and this is the aim of a study on proteins.
Slide 15 In homozygosis, the same mutation gives rise to the same polypeptide chain, so we have only one type of homodimer. For the homodimer it is easier to make severity predictions and suggest or corroborate therapeutic approaches. For example, we can see in homodimers we have to deal with only one protein species. Those modifications that cause a problem in the binding of the co factor can be helped with a high supply of co factor, that I know it is supplied for patients. Up to now the medical treatments are the same for almost all patients, this is why science and research is aiming towards a more precision therapy, personalised therapy because each patient has his mutations and each mutation is different from the other. The problem in compound heterozygosis is that we have two polypeptide chains and three possible combinations of AADC dimers. Two homodimers, each one carrying one mutation and the heterodimer. We are publishing a paper on two heterodimeric proteins where we found a discrepancy between the biochemical features of the heterodimer and the clinical symptoms of the heterozygous patients. This has some sense because the heterozygous patient carries the heterodimer but also the two homodimers, so in this paper, in this investigation, we have proposed that the mutation can complement positively or negatively. Page 13
What does it mean to complement? When we have two different mutations and we see that the activity of the heterodimer is higher than the average of the two homodimers, we say that the complementation is positive. If the activity is lower we say that the complementation is negative. We have found that complementation in heterodimers has a rationale. We are studying and understanding the factors that are controlling dominance. For example, if of the two mutations one gives rise to a protein that is insoluble, many mutations on the surface give rise to aggregate, this is reflected in the extent of the expression of soluble proteins also in the heterodimer. Then the impact of the substitution of amino acid. Since the two active sites are here, and if one mutation is here and the other is here, this will have no impact on the active site. But if one mutation is here, and the other is here, the two different mutations have impact on both active sites, so normally complementation is negative. Why, if they impact only one active site complementation is positive? If one homodimer is highly compromised the clinical phenotype is severe even if the heterodimer can show positive complementation. The activity of the two heterodimers in the compound heterozygous patient is really important. We must not only consider the heterodimeric protein. I can understand this is rather complex. The problem is that complexity is given by the fact that in compound heterozygosis we have three different AADC protein populations and dependant on the type of mutation on the homozygous 1 and 2, and the heterodimer 1 and 2, we can have different results. This is the reason why a compound heterozygous shows a really wide array of symptoms. The population of AADC protein is really large but we have a found a method, a biochemical approach, to study compound heterozygous and analyse the effect of all species in the protein population. We also want to do it with the healthy carriers because this is very important to understand allele dominance.
Slide 16 Another question is can we predict on the basis of the type of mutation whether a child would be suitable for gene therapy or if they will respond better to another child. We have reported some results of therapy prediction. I will show you a really Page 14
optimistic example below, that can validate or orientate drug treatment. But the question about gene therapy for a moderate phenotype, this means the phenotype that produces some protein that is active, is hard to answer up til now. If we perfuse with healthy AADC, the idea is that we generate a condition similar to that of the healthy carrier, the parents, that possess one healthy allele and one affected. So the general vision is that for a moderate phenotype, gene therapy approach would possibly ameliorate because we give some wild type protein. But we do not know anything about dominance. We do not know if we put into the cells this healthy AADC, this polypeptide chain would be dominant over the still producing, still forming chains, that are present in the patients. For severely compromised patients that do not make AADC this is certain of a beneficial outcome. Up to now we don't have enough data to explain, we have to understand better the allele dominance effect. One thing in gene therapy treatment is that patients are not clustered for genotype, protein features. In some records we have no information on the genotype so the results we see, the ameliorating results, are not matched with the genotype at the start and so the protein features. So I cannot understand deeply this question because we do not have, and gene therapy started years ago, not so much, we do not have the reports explaining if some milestones in development and symptoms have been ameliorated for a moderate phenotype. We know for the Taiwanese patients that, and for other patients that have undergone gene therapy, there is an amelioration of conditions. The idea is that they resemble the phenotype of the parents, because you give them some healthy AADC. Recently, in collaboration with Lisa, we tried to combine dopamine and serotonin research because the enzyme producing dopamine, is always studied, but the level of serotonin is not so much studied. The reason is that we cannot exactly match genotype and phenotype to unique symptoms, so we are evaluating serotonin produced in the already published variants, to determine that from a protein point of view the ratio of dopamine serotonin is significant. If dopamine is decreased, but serotonin is made as the healthy protein, this can tell us something about mood control in patients. This could explain why some patients have more mood variations than others, and other non-systemic symptoms that serotonin can exert. We are redetermining for all patients, for all proteins carried by the patients, the serotonin levels, in order to match them to the dopamine levels, to see if there are some differences. This could lead to interesting suggestions in treating Page 15
patients and understanding their mutation.
Slide 17 Another reason why we cannot yet match genotype and phenotype with unique disease symptoms is the individual variability. This is AADC, and this is the reaction played by AADC in producing dopamine. You can see that this reaction is in a pathway involving other important molecules such as noradrenaline, adrenaline and tyrosine. Each one of us has some differences in producing each of these molecules, so if you add to the symptoms of the patients the individual variability, you can understand why it is not so easy to match. It's like the response to a vaccine for example, each one of us has hopefully received a vaccine, some of us has had fever, some not, on which basis it is not known. We generally say the immune system is responding well or not so well and so on. But the disease symptoms are governed, not only by AADC damage but also by a plethory of other molecules that are made in the pathway of dopamine and also in the pathway of serotonin. So the message is we are working to understand as much of the symptoms as possible, but to do this, for example we have obtained a few days ago, tyrosine hydroxylase, and we would like to see if there are some correlations between these two proteins in the wild type and also in the mutated variants. This will be very interesting because this can give us the idea that in some variants the problem is not only the malfunction itself, but also the delivery from the previous enzyme to the forward enzyme. This is really complex and we have a neuronal cell model that will be Crispr in a few days. We are performing now these experiments to try not only in vitro, in solution, but also in cell.
Slide 18 Here I would like to show you some possibilities and hopes that are opening. I have already been speaking with my colleagues on iPSC stem cell that are taken from fibroblast patients. Here we have a publication of two patients, one carries a homozygous mutation, that leads to a catalytic defect into the active site. This is a chemical modification of one important residue at the active site. This is a compound heterozygous which has a deletion of the amino acid, so no protein and makes only this protein, so it is hemizygous. The defect is mild and it is positioned near the substrate site. We have determined that this protein has a Page 16
decreased affinity for L-Dopa and so, in collaboration with the group in the UK, Professor Manju Kurian and Professor Simon Heales, you will hear from tomorrow, suggested L-Dopa therapies to these cells from the patients and in mutation 2 function, that is the treated cells of the patient are responsive to the L-Dopa. The problem of this mutation is a catalytic defect and we can act on this with other molecules, the so called chaperones, molecules that help bypass the catalytic step. This is an example that can give some suggestions for therapy for patients and I know that from cells to patient there is an ocean of problems, but there can be a possible road to improve therapy for such types of patient.
Slide 19 This is my group. I would like to show you the picture of Giada who is now in London performing her research on AADC. Giovanni is doing really hard work to compare on the protein to correlate severity of symptoms to the type of structural and functional modification. We have really important and hopeful results. Thanks to Lisa for the support and the friendship and the AADC Research Trust.
Q&A Julie Are there certain drugs that respond better to certain mutations?
Professor Mita Bertoldi The treatment up to now is based on a triad of three types of treatment. Pyridoxine, that is the supply for the co-enzyme because without the co-enzyme the enzyme does not function. Dopamine agonist because the enzyme is not producing enough dopamine and so the agonist lets the dopamine receptor transmit the message. And then monoamine oxidase inhibitors, because normally the dopamine is destroyed by monoamine oxidase physiologically, and if we give inhibitors we will prevent the little amount of dopamine being destroyed. So the election treatment is this one. In this sense all three components with others are essential for the therapy. It is not a question of which functions better but inside the dopamine agonist, patients experimented that with one dopamine agonist the response is better than with the other and this is in the field based of individual sensitivity. Page 17
It is nothing to do with AADC, because dopamine agonist interacts with the dopamine receptor, so the problem is not related to AADC. With AADC a possible treatment is L-Dopa but only if our biochemical data showed there is a problem in Dopa binding otherwise it is not useful. A greater amount of pyridoxine is important if the mutation touches some residues in the PLP binding site, so the PLP is not binding well. Some chaperones could be important. We are now starting a set of experiments because we have understood that the interface residues between the two monomers are really important and we wanted to address some chaperones to make the interface be more intertwined in order to allow the enzyme to function better. The variability of treatment in the same set of agonists, or treatment, or drugs, is individual. It's not related to mutations.
Julie Do you think gene therapy would be best for my one year old son who is of moderate phenotype or should he just stay on medication?
Professor Mita Bertoldi I am a biochemist and not a doctor or clinician. I am basing only on scientific results. I think that the gene therapy that Krys is performing is really oriented to a part of the brain that perfuses dopamine in a more aimed manner. But I think if the phenotype is severe then gene therapy is an almost necessary treatment. But I cannot say, there is no research, but when I have more data, more published data on output, on the results of people treated with gene therapy, and this means after a couple of years because we have to see the progression of neurotransmitters on a large timescale, this will give us more rational data. On a very personal basis I think that gene therapy could be helpful for very young infants because it prevents that damage of the brain after years of experiment of children, after years of decreased dopamine and serotonin. I also understand the compassionate gene therapy to older people. I can share my suggestion to take gene therapy but I have no scientific data up to now. We hope to have them in a short time but for now, with my expertise, I cannot say anything. I am very sorry but I cannot say anything as I have no rational basis. Page 18
Julie After gene therapy the biochemical results show an increase in dopamine not serotonin, do you think families should consider treating the serotonin deficiency with SSRIs?
Professor Mita Bertoldi I would say from what is published in literature, not so much is known about 5-hydroxytrytophan and the agonist of serotonin. I know that since serotonin effects more psychiatric symptoms than motor symptoms, we have to perform more research on the cell model to understand that if there is a real benefit. If this is really beneficial because I think that an overlap with dopamine agonist and SSRi, could be, we have not enough data to answer, but I know that some research is ongoing. The problem is that this disease has been neglected for several years. Research has begun really only 10 years ago, real basic research. It is one thing to counteract symptoms as a medical doctor, which you can do with success, but it is another thing to find the molecular cause of the defect and counteract the molecular cause. This is really new and so we do not have much data but we are really working on this to fill the gap.
Julie How do you determine whether a child with AADC may be a candidate for and respond better to L-Dopa?
Professor Mita Bertoldi I can measure a parameter, that is the technical name, and this parameter is called Km and is a constant. We measure with our kinetic analysis, and this parameter gives us information of the affinity of dopa for the enzyme. Affinity means avidity, how dopa is able and binds to the enzyme. We know that in healthy protein dopa binds really healthy in nanomolar range and in some mutations dopa binds in millimolar range, so not so much because we need millimolar quantities to make dopa bind to the enzyme. So we measure and we find if this variant, if this mutation is a candidate for dopa treatment. We did it and we are doing it.
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Lisa Can you determine L-Dopa possible response in heterozygo, homozygo and hemizygo?
Professor Mita Bertoldi Yes.
Lisa So can you do that when you don't know the dominance as well? For all combinations you can do an L-Dopa analysis?
Professor Mita Bertoldi As long as I have the enzyme I can do it. I can give the enzyme, the L-Dopa, and measure the rate of production and I put these points on the plot and from this, in a mathematical point of view, I can extract the meaning of affinity, the avidity of that enzyme, homodimer, heterodimer for L-Dopa.
Lisa From the list of combinations of mutations that you showed us earlier can you determine from those who you would predict the L-Dopa would work?
Professor Mita Bertoldi We have determined this parameter for many of these mutations. There are published data and we are determining for many of these, I have just submitted a paper for publication, on heterozygosis, on two heterozygosis mutations. Each one has three types of proteins and I gave a rationale for affinity for the substrate and for chemical activity. We are able to propose to see if the effects of the protein malfunctioning are due to bad dopa binding and so this is a prerequisite to be a candidate to L-Dopa treatment. For example, all mutations at the active site have a problem with dopa binding. Interface mutations, some of them, have a problem with dopa binding.
Lisa For simplicity, that list of combined mutations, if we were to know which ones you could predict would respond to L-Dopa, just in a simple table most of our families Page 20
will know what their mutation combinations are so they can identify, and then they can talk to their doctors about possible treatment.
Professor Mita Bertoldi, If you send me the combinations of mutations of the parents that are interested I can tell you if we have already characterised this parameter or if we will characterise it.
Lisa And the combination of L-Dopa and dopamine agonist. Would you know if they'd respond better?
Professor Mita Bertoldi I don't know from a medical point of view what is the output of combining L-Dopa with dopamine agonist because it is the same. But they function on two different systems. One increases dopamine. The other occupies the receptor, I do not know if there's some cross work and some competition is made.
Julie Crispr KAS 9 and Crisp ON, have been featured in the news a lot lately, is this treatment actually close to being an option and do you think AADC may be cured by it in the future?
Professor Mita Bertoldi This question is really important but up to now we have succeeded in a Crispr single gene in simple cell models. I think that for the future it is really interesting because you are silencing the diseased gene, and put inside the cell a really functioning AADC gene. This is easy from a theoretical point of view. It is almost easy, in some simple cell models, like neuronal cells, but the passage through human cells and then to organisms is a bit more difficult. I think every strategy that can counteract this rare type of disease is to be taken into consideration but we need to perform some basic research before. I think for the future it can be, but for now we should go step by step.
Julie We heard about promising studies on intestinal bacteria/probiotics increasing Page 21
AADC activity. What is the mechanism?
Professor Mita Bertoldi Intestinal bacteria have their own AADC, so the mechanism is that they supply with the peripheral AADC. This is very important. We are thinking to try enzyme replacement therapy to target the kidney, because to target the brain is really difficult, to over pass the blood brain barrier. We think this could be a tentative way to start, to help patients. The answer is it is important because it increases dopamine but peripheral dopamine, and the problem of the children is brain dopamine. So it may be useful in some ways as in the kidney, most of the production of epinephrine and norepinephrine is peripheral, so it can help in increasing this neurotransmitters extent, but it should be combined with other approaches. This is really really interesting.
Julie Could protein foods higher in amino acids be beneficial?
Professor Mita Bertoldi Protein foods that contain precursors of dopamine and serotonin and this means animal protein. Protein of animal origin. Sorry for the vegetarians, they have to supply by pills to get the same. Animal proteins contain a lot of, for example, a great amount of tryptophan that is not contained in vegetable proteins, not so much. One should eat a lot of beans, like English people, who eat beans in the morning like me. I discovered it in the UK, but I love it too much. Animal protein is a source. Also milk is particularly rich with tryptophan. Drinking a glass of milk in the evening that is full of tryptophan, which is converted by our enzyme, 5-hydroxytryptophan, is converted to serotonin which is the neurotransmitter of serenity, calm, joy and the sleeping facility. So animal proteins, milk, we have to supply with tryptophan and phenylananine or tyrosine from meat and so on. Vegetables contain these essential amino acids, because phenylananine and tryptophan are essential. We produce these amino acids but not in enough quantities. So if one is a vegetarian one has to supply with pharmacy and so on this supply of essential amino acids.
Julie We find that supplementing Forskolin Cyclic AMP improves our sons symptoms. He is compound heterozygous. Would you be so kind to share your thoughts on cyclic AMP involvement in acid enzyme activity? Page 22
Professor Mita Bertoldi Cyclic AMP is a regulator molecule. We do not know the exact linkage between cyclic AMP and AADC but cyclic AMP, one of its function is to activate enzymes that are mediating phosphorylation. Phosphorylation is a mechanism of the regulation of the activity of many enzymes. We do not know anything about phosphorylation of AADC but our preliminary data of AADC in solution showed that, one of my PhD students went to the United States, and studied phosphorylation of AADC and found some serine residues at the active site that are phosphorylated. So it is a possible effect but I have no scientific basis of beneficial effect of cyclic AMP is to get the phosphorylation levels higher, and phosphorylation of AADC has no effect on chemical reaction, in the velocity of dopamine and serotonin production, but it seems that it makes dopa and tryptophan more able, it increases the affinity of dopa and tryptophan to bind to AADC. The parameter of that Lisa asked me before. The phosphorylation of AADC in some residues of in the active site, seems to ameliorate dopa and tryptophan binding, thus dopamine and serotonin. I can think that in some variants, that are not so efficient in binding dopa and dopamine, it could be important. It could have some effect but up to now my only results are in vitro. I do not know if it happens in cells. In vitro, phosphorylation enhances affinity for the substrates, so it could be the effect on the child is due to this amelioration in the regulation of phosphorylation. This is another topic of our research. We would like to know if this phosphoryilation we observed in the wild type protein, exerts the same effect on others in the pathogenic variants.
Lisa Would Bovine adrenal gland cortex extract in powder form also increase peripheral norepinephrine and epinephrine in AADC kids so that they can better manage daily stress levels?
Professor Mita Bertoldi I don't really know because it is a more medical question. I think that the adrenal gland cortex extract can be beneficial in raising the levels of epinephrine or norepinephrine but it is really outside of my expertise. From a rational point of view, I don't know the counter effect or cross effect. It can be there is a rationale.
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Lisa In Crohn's disease, which greatly impacts the gut, a Faecal Microbiota Transplant (FMT) is sometimes facilitated to improve symptoms. Do you think something like this may be an option for AADC patients to improve gut function and increase AADC production?
Professor Mita Bertoldi This is another question that I do not know because I am studying the direct correlation with AADC. I can look at the literature and see if there is some interaction between this and the pathway of dopamine and serotonin synthesis. If I see that there are some molecules involved in the pathway, this could be but I don't know.
Lisa I think the PS128 that Dr Lee was talking about might help, so we might be able to put that question back to Dr Lee.
Professor Mita Bertoldi I hope I have been clear and not spoken too rapidly. This is more technical. If you have more questions that come to you in the next few days you can transfer them to me. It is a pleasure to answer and contribute, from my point of view, in this struggle against AADC deficiency.
Lisa I have one more question. In carriers, particularly the hemizygous and homozygous could you predict whether they would be symptomatic carriers and recommend any treatment? Heterozygous is more complicated because of dominance but in the hemizygous and homozygous would you expect them to have symptoms and could they potentially be treated?
Professor Mita Bertoldi I think that these types of people are really interesting from a research point of view. We don't really know, we do not have great news on these, the hemizygous, the carriers. What we are doing now, we have the system function, is to try to mimic the carrier genotype in order to understand if possible symptoms that are not so severe can be due to the other allele the mutated one. And to give some suggestions. To have a molecular basis to give a suggestions, but right now we do Page 24
not have it.
Lisa It has been a pleasure Mita thank you. Brilliant presentation. I know that everybody will watch it back and study what you've said and there will be questions I am sure. Please carry on the great work. The genetic understanding is so important to personalise medicine for each of these children who are presenting differently. It is good to learn more about that. So thank you very much for today and the extra time you have taken.
Professor Mita Bertoldi It is always a pleasure for me and for you all parents. I really miss the meetings in London. It was really nice to meet the families and children. And to speak to each other and understand what the point is. I found that the parents are very informed about scientific matters so it was really important and informative for me too.
Lisa Thank you. And thanks everybody for joining today and hopefully we will see you tomorrow for our final one. Have a good morning, afternoon or evening wherever you are.
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AADCd Family Workshop 4 : Transcript Produced by Julie Ramsay Verified by Lisa Flint & Professor Mita Bertoldi The AADC Research Trust would like to thank all involved.
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