tion sequencing data, we're learning more and more about the gene. But there is one very common mutation that affects the binding of LDL receptor to Applebee to LDL cholesterol. It affects about 6 to 10% of all FH mutations. It accounts for 6 to 10% of all FH mutations, but it's just like in my golf phenotype. And again, we will look at that a little bit later. Gain of function mutation in the PCSK9. Again, this gene is difficult to study because you need to distinguish between gain of function and loss of function. So that requires functional work studies, cell work, for example. But that mutation is very rare, affects about 2% of FH cases, and however leads to most severe cause of hyperchrystalline and highest risk of premature coronary heart disease. So important to look for it. This is kind of the proportions of these three genes in terms of the mutations, mutations in the ones that lead to recessive FH are relatively rare. So well, again, we will not be covering that. So this is the new gene here. The frequency overall, it's not known yet. It's something that we are working on at the moment. We think it's higher than the mutation in PCSK9. But again, the phenotype is milder. So it will be something more like the Applebee mutation. But there's plenty of different variants that cause FH. Last time I looked, there was over 1,700 different monogenic causes of FH reported worldwide. Here are two main mutation databases for FH. I will show you a little bit more about that later. But the bottom line is that the cause of FH is highly heterogeneous, so many different causes. There are some populations, however, where just few mutations account for the majority of FH. For example, in Greece, Finland or French, Canadian. It is important to know that if you want to design an effective genetic testing for FH. So that you know what methods to use, how to adapt it. I think I have a question here. So how frequent is FH? I think 10 years ago if someone asked me how frequent is FH, we would classify FH as a rare disease under the definition that we use here in Europe. And it was the old sort of estimated frequency of heterozygous mutations was 1 in 500. So 1 in 500 individuals in a population would inherit a mutation that leads to FH. But recent studies, genetic studies based on next generation sequencing methodologies, discovered or confirmed more and more variants. And so the frequency now we estimate is about 1 in 250 or 1 in 300. So suddenly the frequency kind of doubled. And this is because we have much better methods now to test the mutations. And in the previous era, let's say it was only an estimation. So how about homozygotophage. So this is when both parents are affected, they both have an FH mutation. And so when they have children, they have 25% chance that their offspring will inherit both of those faulty variants. And so that will result in homozygotophage. Homozygotophage, it's a severe case. It's children at the age of 5, 10 year olds develop early, develop cardiovascular problems. They require very specific treatment. And I'll show you a little bit in the next part of my talk about that. So it's in a way a tragic event. And we used to think that it affects one in a million individuals, but again, genetic studies suggest that actually it is much more common. It's more like 1 in 300,000 individuals who have homozygotophage. So in the UK, we estimated that we've got about 200 cases of homozygotophage children. And actually, we don't know at the moment where they are. So we're still kind of behind with diagnosing them, which is quite astonishing because I think that the phenotype is very, very obvious. But obviously not many people test the cholesterol level in children. We will again discover a little bit more about that later. But yes, so it isn't a rare disease. It affects more individuals than we previously thought. And there are even populations where FH is even more common. 1 in 100 has been estimated to affect French Canadians, Christian Lebanese or South African African Africaners or Ashkenazi Jewish individuals. So these are sort of genetic isolates. And it can go the other way. So for example, in Iceland, also a genetically isolated population. A recent report using a negative generation sequencing data suggests that FH frequency was 1 in 800. So they lucky they've got much less FH in their population. But I think the bottom line is that FH remains highly underdiagnosed. So this is a figure from 2013. That shows you the percentage of individuals diagnosed with FH in each of those countries where we had data for. So Netherlands are doing very well. They had 71% of FH cases identified. They had very well implemented cascade testing program where you trace families who are affected and then you identify all the affected individuals. And then you can go and expand your search going through another generation. And so they've been doing very well. But imagine this is actually based on the previous frequency, the historical frequency of 1 in 500. So if you think that actually mutations affect 1 in 250, you will need to divide all those numbers by two. So we're not doing very well at all. Not even the Netherlands. UK as well. Our figure was here 12%. I think since 2013, we are getting better. So if you divide that number, we probably have about 7 to 10% of FH cases currently identified. So there is loads to be done still. Those individuals are walking around not knowing that they are affected. And more needs to be done to prevent the heart attack. So then the question arises, how can we systematically identify FH individuals? And why is this very important? Why do we have to put loads of effort into that? And first to answer that question, I think we need to look at the clinical diagnosis of FH. So how can you see that someone has FH? And the major criteria is cholesterol measurement, of course. So we've got a total cholesterol of 7.5 millimole per litre or LDL cholesterol of 4.9 millimole per litre cutoff in adults. This is using the Simon-Brom criteria, which is one of the main defined criteria to identify FH. To look at children, the cutoffs are slightly lower. If you remember on one of my first slides, family history is very important to analyse family history. There is a clear pattern of autosomal dominant inheritance that strongly suggests it can be familial hypercholesteroidemia. So if you have the high cholesterol and family history of either high cholesterol or premature heart disease, then that will classify a patient as possible FH. Now, in addition, if an individual has tendon somatomas, so this is kind of like a physical deposition of cholesterol in the tissue. It can be observed around ankles, some people have around eyes. That classifies patient as definite FH. So there's clearly something going on. There is a high burden of cholesterol. Those individuals have been exposed to high cholesterol levels for many years, and so we've got to build up. Also, genetic testing is in place and we will talk much more about it in the next few slides. If a patient has a FH mutation, obviously that gives them definite FH diagnosis. Worldwide, there are two different systems as well. We've got the Dutch scoring system to define FH and there is a in the US med-pad criteria. All of them roughly perform in the same way in terms of identifying patients who carry an FH mutation. And now about 40% of those possible FH cases and 80% of definite FH cases actually have an FH mutation in the known genes. So clearly there is something else going on. We're missing about 60% and the second part of my talk will be kind of explaining why this is the case. So I think that suggests really that those criteria, although they were not bad, they are not perfect. So there is perhaps other ways that we can improve the criteria in order to identify those individuals who have an FH mutation. So we know the clinical diagnosis of FH, so now why is it important to identify those individuals early? And this is because patients have premature heart disease. So why do they have heart disease so early? And this is because of the LDL cholesterol burden. It has been estimated that 50% of men and 30% of women will get CHD by the age of 55 years. Here is a plot of that burden, so LDL cholesterol exposure in terms of multiplied by the years. So how long individuals have been sort of exposed to high cholesterol levels. And I said earlier to you that it happens from birth really FH mutation carriers have higher LDL cholesterol than non-mutation carriers. And so here is a plot of a FH individual in blue, in red, non-FH individual. He may say first of all, they're not really different that much, but if you think about looking into a 45-year-old individual with a FH, their burden of cholesterol is as much as about 70, 72-year-old healthy individual. So really there is a big difference in terms of age. They are exposed to high cholesterol much earlier and the burden is much more severe. And there is a need really for aggressive treatment to reduce that burden. And that treatment really should be started early in childhood. The current recommendations say to start starting treatment in FH children by the age of eight years. So yes, and the early diagnosis and treatment will give you a good chance to prevent CHD. So you may say, okay, well, why don't we just screen using cholesterol levels, check cholesterol level in the whole population and then we all know who is affected. That would be nice in a way a cheap method to do that. But the problem is that there is an overlap in terms of cholesterol distribution. So here is a histogram for non-FH individuals in blue and a FH mutation carries in red for children age 5 to 15-year-olds. And you can clearly see that those FH individuals have higher median cholesterol levels than those unaffected, but you will also see that overlap here. And that's the problem when we struggle with the diagnosis in those individuals. So using a cut of somewhere in between here between those two overlapping curves would give us 8% of the actual individuals who don't have a mutation being classified as a FH. But I think what is the bigger problem is the false negative rate. So 15% of individuals who have actually FH mutation would not be identified using just LDL cholesterol measures. And that is an issue that actually increases with age. So here is the same graph for 45 to 54-year-olds. And you can see that the overlap between affected and affected is massive. And that would lead, if we again take the cutoff sort of in the middle, that would lead to very significant false negative. So you would miss basically 46% of affected individuals if you were to use cholesterol levels as the sort of diagnostic tool. So as mean LDL cholesterol rises with age in every single one of us, the non-FH and FH distribution overlaps. And so DNA testing would be a good solution here to say who is actually carrying an FH mutation and who isn't. So let's have a look at the genetic testing. This is something that is very useful tool, it gives you a crucial information for some screening strategies which we will discuss later. If you really are into this kind of methodologies, we have recently published this review in the Journal of Lipid Research. It's quite a long review where you can find a bit more about the methods that were used in the past that are used currently and that perhaps will be applied in the future in terms of identifying FH cases. So let's have a briefly look at those methods. In the past, we had methods that were really not specific but there was no human genome published, we were not aware of the specific sequence of the genes that were identified to cause FH. So it was, there was loads of information missing. But one of the methods to identify and what allele mutation that causes FH may be located was restriction fragment length polymorphism or RFLP, which is a cutting a fragment of DNA with a site-specific restriction enzymes. So restriction enzymes come from bacteria and they are sort of a preventive mechanism to destroy viral DNA. And so they cut DNA at specific sites and we know the sites and we could, if you know of polymorphisms that occur in your gene, you might want to use that kind of technology. So what to use to apply that methodology in terms of identifying disease, you require rather large family tree and many samples to be tested to decipher who actually carries, who inherits the mutation. So that in this case, we have an example of the restriction enzyme called PVU2 that cuts at a specific site. It either cuts if you have the variant or it doesn't cut it and you get sort of, you can see different size of the bands on sudden blood. So in terms of this family, you had the problem here, we can, after analyzing, you can see that the phenotype, FH phenotype co-segregates with the V1 allele. But the specific variant is not identified. So we can then go further. Now we've got the technologies to sequence DNA very quickly and cheaply, but previously that kind of methodology was used. So the index case in this case needs to be heterozygous. It doesn't work for homozygous cases. It needs to be heterozygous for the restriction enzyme cutting site. It doesn't identify, as I said, specific mutation but only sort of looks at the inheritance pattern and mode in the family and it requires rather large family tree. So not very effective method. We then moved to different assays. There was ARMS kit, which you could sort of buy ready mixed assay that would test for 20 most common FH mutations. It was a nice test that when I was doing my PhD, I actually applied that. It was still in use, but again, it looks at only 20 mutations. It needs to be sort of specific to the population because you're looking at the most common mutations in your population, and that varies between different countries. And it is only 20 mutations. If you remember from one of my previous slides, we know now of more than 1,700 different cases, different mutations that lead to FH. So if you think of 20, it's really pretty bad. We then had HRM, the high resolution melting, and here is a lovely curve of that. I think Professor Ali Akta used that method in our lab when he was doing his PhD. So you may want to ask him for details, but it's a lovely method based on PCR that you would be looking at different melting curves. So you make your PCR fragment and then you melt it. And because if a person has a variant, that affects the melting temperature. So here is the temperature on this axis. And you can clearly see these individuals in blue are unaffected and the red ones actually carry a mutation. So again, it doesn't tell you what exactly the mutation is, but you can see the difference. And then you can take those individuals and sequence the fragment of the DNA to identify the specific variant. It was, as I said, a lovely method, but quite laborious actually. You need to divide your DNA into chunks and then do it over and over for every exon of the gene. So yes, there are still some limitations to that. Another assay MLPA, multiplex ligation, dependent probe amplification, important assay for detection of large rearrangements within the gene. Again, this can also be done these days by next generation sequencing. So this methods were definitely more accurate than the RFLP, but they are still very laborious. They can be costly as well and there's multiple steps that are required. And so you've got multiple steps in the lab that leads to higher chances for human error, unfortunately. So some things could be missed. So what is the present really in terms of genetic testing for a ph? It is the next generation sequencing methods, more precisely targeted sequencing. So here is an assay where we target, we select fragments of the known genes from the whole genome and genes that are known to be to associate with a ph. And they are all the libraries prepared. So you pull out all those regions from LDLR, Applebee, PCSK9, you can put in AppleE, you can put any other novel gene that you identify. And they are all sequenced at the same time in one assay. So much less time required. There is less room for an error. And you can also, as I said in the previous slide, detect the large rearrangements within the gene when you plot the coverage, so the read depth of the of the essence. Here is an example. And again, if you want to read a little bit more, you can go to this paper here. It's an example how you can use the next generation sequencing data to identify, in this case, it's a deletion of exons three, four and five and six. You can see the read depth is much lower than the average. And that suggests strongly that there is a deletion of those exons in this, in this patient in LDL receptor gene. There are still unfortunately some limitations to those methods. And these are mainly due to genetic variation interpretation. So we are identifying loads of novel variants, and we are not quite sure how to interpret them. Again, functional studies or family co segregation is important to prove that a specific variant leads to the phenotype. And it also requires specific bioinformatics skills, which still there is seem to be a shortage worldwide. So how about the future. I think next generation sequencing is here to stay for sure. And we are moving, if you have loads of money, we are moving towards whole exon or whole genome sequencing. And in that case, you can look at not only the reverence, but also common variations so SNPs polymorphisms. And that leads me to the polygenic risk scores and we will discuss the polygenic cause of a phage later on. But that method looks at the whole genome. So it's not only selecting specific genes that cause a fight. You can find all sorts of stuff to be honest and. And that's why because we're finding so much. One of the biggest issues that again interpretation of the data. Plus, the methods are still relatively expensive. And you also require high performance computing, in addition to the bioinformatics skills that are required. And what else we need to do is to ensure that we have the reference population data that is diverse. There is an unfortunate in genetic studies still bias towards individuals of European background. And so most of the databases that we can use as our controls or our reference come from individuals of white European background. Therefore, if we are studying different population. There is a clear discrepancies if you identify a rare variant. You may identify a rare variant that is not present in the Europeans, but perhaps is very common in in South Asians. So. One may get excited that we identify novel rare variant in in I don't know in Germany. But then you go and look at a large genetic database in India, for example, and you'll find that that variant is actually quite common in that population and it doesn't associate with any phenotype. So there is a lot of effort at the moment going into that to make the reference genome much more diverse. That's something that we definitely need. Okay, so let's come back to to Mike's family. So again, if you remember, Mike had a heart attack at the age of 35. And so after looking at his cholesterol levels, the doctors referred him for genetic testing for FH. And so he was his DNA sample was sent for targeted sequencing. So this is the slide where I show you the present state of art DNA testing for FH. His DNA was sequenced for the LDL receptor for Applebee and PCSK9. And after, I don't know, I think the turnaround is about three weeks. The data were analyzed and what was identified is that Mike has an LDL R mutation. It's a premature stop codon formation. So the star indicates that there is a stop codon. So this is the cysteine 231 amino acid that one base pair leads to premature stop codes that basically the gene terminates at that position. This is relatively early in the gene. LDL receptor gene has 862 if I remember well amino acids. So clearly if you cut it at 231, you're missing big chunk of the gene. So it's a null mutation in a way. The question then was, okay, we did the targeted sequencing on Mike. The question is obviously do his children, can his children be affected? This is very important question that family needs to consider. Do they want to know? They should know, I think, in terms of we've got treatment for the children. There is loads of studies showing that introducing treatment at early age can prevent heart disease. So very important question. And that would be sort of part of cascade testing. These two little people are first degree relatives of an individual who has an effect mutation. It is very fair to look at their DNA. Now, because we know Mike's specific mutation, we don't have to then pay for expensive targeted sequencing, but we can only look if they inherit this specific mutation. So a cellular sequencing of a specific fragment of the gene can be applied for a quick test. And that's what happened. And unfortunately, Daniel has been identified to also inherit the mutation. I think this could have been concluded from the fact that he has a higher lipid level than his mum and he's relatively young. But if you think about the overlap in the cholesterol distribution, it could go really both ways. The DNA testing really gives him accurate results here. On the other hand, Matthew tested negative. So here, big sigh, he's relieved he has not inherited the disease. So I suppose this is very positive news for him. In a way, I think for Daniel, it is positive because now he knows he can adapt his lifestyle, he can have a treatment and he can prevent the disease. Obviously, mum was not tested because she's not genetically related to Mike and her cholesterol levels are within normal range for an adult. So yes, this is how you can implement DNA testing to screen family. So how about mutation interpretation? I think I've looked through your program and I've got a feeling that you might have already talked about it. I have mentioned to you before that this still remains a significant limitation in terms of DNA testing for a phage and more efforts to prove that a specific genetic variant causes a phage are needed, especially for variants of unknown significance. So since we have the next generation sequencing and we are being screening through all the sequences of the three genes, what we identify is that a significant proportion of the variants we cannot say if they cause or not cause the disease. So this is important from genetic counseling point of view. So imagine that we found a view where so variant of unknown significance in this family telling them that we don't have enough information to prove that there are variant identify causes of age. So it leaves them sort of in limbo. They don't know if Daniel is affected. They don't know what to do really. So I think having diagnosis is very important for family. It gives them, you know, you can then action basically and you know what to in a way to expect. So sorting out those variants of unknown significance is important. And here is a graph of the clean vase and this is another database that collects all variants associated with different phenotypes. So here in specifically we looked at variants that affect that are associated with a phage and benign are the yellow ones. These are not affecting so not pathogenic variants. The pathogenic are the ones in red and the view us is the variants of unknown significance are in blue. There's still another group of unclear, which I think are because of the wrong submission to the CleanVar database. CleanVar is a sort of open access database where anyone who finds a variant can then submit it to that database. And believe me or not, many people would submit things that don't make make sense. For example, wrong base pair or wrong amino acid number and we know the standard sequence of LDL receptor, for example. So if someone is submitting something that doesn't make sense, we can't really analyze it. So yes, majority variants in LDLR I think we're doing very well in terms of interpreting the variants. We know a lot about the gene, about its structure, about its function. And so majority would be classified as pathogenic variants. We've got only eight here, percent of LDLR variant being difficult to interpret or uncertain. In terms of apoB it's a different story and it's the same for the PCSK9. So I said these two genes are still understudied, especially apoB. It's a very large gene with loads of different variants, loads of rare variants. And so majority of the variants that we find in apoB are variants of unknown significance. So very important to find an assay that can define the effect of those variants on LDL cholesterol. And PCSK9, again, it's a similar issue when you have to distinguish between loss of function and the gain of function variants. So family studies are a good way to analyze the effect of variants. So if the variant clearly co-segregates with the family phenotype, with a fade phenotype in a family, then we can see that that gives you a strong evidence that, yes, perhaps that variant really truly causes a fade. A large databases, again, from what's important diverse populations to establish frequency of those variants is also important. And these are, again, the databases that are gene-specific and curated. So this is people who are really experts in the function of, for example, LDL receptor. We had Sarah Lee, Mofau de Bourbon, who are people who really sit down and analyze variant by variant and decide which variant is pathogenic and which one is not. There's loads of different criteria that you need to take to define a variant as a pathogenic. To avoid sort of false diagnosis. Those criteria have been defined, for example, by the Association for Clinical Genomics Science back in 2020. So you might want to follow those guidelines to classify a variant. If you go to these databases, these guidelines have been already taken into consideration. Not on CLEANVA, unfortunately. So CLEANVA is kind of, it's great thing to see what people find. But as I said, the interpretation can be a little bit subjective. So you don't know if the person who is actually submitting the variant actually have gone and read those guidelines. Yeah, it's a big job to do, to interpret the variants. You don't want to give a patient false positive diagnosis, for example. If you find a variant, because that's not right. And it's the same with false negative, giving someone a diagnosis, you don't have an FH mutation, you can go away and just look after your diet. It's also not right. So this is important part of your genetic diagnosis. So why does it really matter knowing specific mutation? We know that a patient has high cholesterol, we know they've got a mutation. And why do I want to know if they've got mutation LDLR, POP, CSK9? Well, I think it has some value for sure. First of all, we observed that the risk of premature coronary heart disease differs by different phenotype or different gene mutated. And the PCSK9 mutation carriers have the highest LDL cholesterol and they also respond to statin treatment quite poorly. And that, I suppose, leads to the high risk of premature CHD. Whereas apoB mutation carriers have quite low cholesterol levels to start with in comparison to other mutation carriers and their risk of CHD is also relatively low. Recently we looked at children with FH mutations. They have a large database of different registries across Europe. And what we were able then to do is to further divide LDLR variants into different classes. So for example, variants reported in the promoter, miscense variants, splicing variants, nonsense and large rearrangement within the gene. And what we found is that the LDL are large insertions and deletions as well as nonsense mutations. So these are the ones that Mike in our example family had. So a variant that leads to premature stop coden, these are one of the most severe ones. So this is when you don't have a functional LDL receptor. So in a way, we are leading towards this kind of precision medicine approach where one can look at a variant and think, All right, I know I need to give this person more aggressive treatment. I need to, for example, see them more often in the clinic, especially if they have a PCS-Q9 mutation, because I know that they might be at a higher risk of CHD. Obviously, defining those findings requires large number of patients to study, but I think we are getting somewhere with it. So, yeah, definitely step towards some precision medicine approach here. Another question that I get asked is, okay, so we have the FH mutation. We know the phenotype, for example, they've got high cholesterol, but again, we have now looking at population data, there is a significant proportion of people who have an FH mutation, but their cholesterol level is actually okay. It doesn't go over the diagnostic threshold defined by Simon Brom criteria, for example. And so what do you do with that individual? Do you treat them? Do you see them in the clinic? I mean, they don't have high cholesterol, so they should be right healthy and they can look after themselves. Well, we need to be very careful with such conclusion. There's more studies showing that the risk of cardiovascular disease is always higher in FH mutation cures, no matter what their cholesterol level is. Here was a study from Cara et al. that showed a difference, so here is a LDL cholesterol, different sort of grouping of LDL cholesterol. This is now given in milligrams per decilitres, a different cut of them, what I presented to you before. But basically those individuals that definitely don't have high cholesterol and these guys are kind of normal cholesterol levels. And you can still see in that dark purple, these are the mutation carriers at any level of cholesterol levels, that mutation carriers have significantly higher risk of, or odds ratios for coronary heart disease. So yeah, it's, I think it's important to know that mutation. And I think what we need to start thinking is that mutation itself, it's in a way an independent risk factor, independent from the cholesterol level. A recent study, very recent, just published in the circulation genomics precision medicine, again suggests that after adjusting for LDL cholesterol levels. The mutation carriers are still significantly higher risk of CHD. So, yes, again, another evidence that CHD is independent in terms of mutation cars, independent from the exposure to LDL cholesterol. Of course, knowing the mutation is also very crucial piece of information for screening strategy and that's what I want to talk to you about now. What can you do, how can you take all that knowledge or mutations on LDL cholesterol distribution or all the overlaps and how can you utilize that information to effectively identify FH individuals. Well, we know that early diagnosis and treatment are very beneficial for FH patients. So there is no doubt, I think that we want to identify those individuals. And of course, preventing cardiovascular events gives significant savings to the national health system through the reduction of those events. They are very costly if a person ends up with a heart attack in the hospital that costs much more than prescribing statins from the age of 10 year old statins are relatively cheap. And so there is no doubt that this prevention early identification before any cardiovascular events is beneficial in terms of money. Now cascade testing, so this is when you trace families using the DNA information is very cost effective. So because it's autosomal dominant disease, you're expected to find 50% of your first degree relatives also being affected and that 50% is relatively high for screening strategy. And so it has been implemented in the UK. And now knowing the family, the family mutation is a key piece of information to make it cost effective. So if you remember again, Mike's family, the next generation sequencing assay was first applied. But then once the mutations identify you can use cheap sangra sequencing to confirm the variant in his relatives. And it's also very important evidence to start early treatment in children. So once there is a mutation, there's no doubt that that child should start thinking about receiving treatment. But if you think about cascade testing, you still need to identify that initial index case to then trace the families. And it is the issue with identifying index cases that we are way behind with. So, again, going back to Mike's family, he had a heart attack that's too late to identify him. That's how he became the index case, we need to make sure that we find index cases way before that. So one of the approaches to identify early individuals who have an FH mutation is child to parent screening. Now this is when it's currently being tested actually in the UK there is a large pilot and I think it's 30,000 children who come to their general practice for MMR vaccination. After having the vaccination, a blood spot sample is taken, and their cholesterol level is measured. And if their cholesterol level is higher than the average, I mean, then it's I think over a 90th percentile, then they are invited those children are invited for DNA test. And then the parents as well are being tested. So obviously, you expect one of the parents of the affected child to have an FH mutation. So we kind of going backwards from a child to a parent rather than from a parent or child. The idea is good, I think we would be identifying those children very early on. There's loads of issues with sort of logistics of it. But as I said, it's currently being tested and it might be at some point implemented. Another way is to opportunistically search through electronic health records that GPs have. So here, you know, test cholesterol levels. In England, we test how every 40 year old individual is invited for sort of general health check and cholesterol levels are then measured at that time. If their cholesterol is above a certain point, then they would be perhaps invited for DNA testing. But this isn't systematic, really. It hasn't been well implemented yet, although it's a nice guidelines. So this is sort of a policy for health care in England recommends doing that. It hasn't been going very well. So we still have the issue of underdiagnosis. Majority of FH cases, unfortunately, remain undiagnosed. So why is it not that easy to detect individuals in a population? Very recent work here by my PhD student, Jasmine Bratton, what we did is we used the UK Biobank resource. And so this is half a million individuals of UK. They are mainly white British, but we've in this study focused only on the white British to make it sort of cleaner. The data for whole exome sequencing is available and it was available at the time of the study for 140,000 individuals. So we looked into the 140,000 individuals of UK Biobank. We extracted DNA information for LDLR, Applebee, PCS-39, we looked at their mutations and we identified among those 488 mutations. So the prevalence of FH mutations in the UK Biobank cohort was about 1 in 288. So relatively close to the 1 in 250. And yeah, and now the question is what cutoff of LDL cholesterol could be used to, for example, screen those individuals to use that information to screen individuals in a population. So if you were to use the 4.9 millimole per liter cutoff, that's the one that Simon Broome criteria uses. We would find almost 40% of individuals who have a mutation and have the phenotype, but we would miss 60%. So 292 roughly individuals who have a mutation would be missed. So in blue you've got the cases missed. And obviously the higher the cholesterol the more cases you identify. But if you are designing a population screening, you need to of course think about all your resources. So what is your point? You will be taking those individuals and sequencing all of them to identify the mutations. So it's really a matter of discussion in terms of, on a country basis, what are your resources? How many people can you screen? How many people can you sequence? What is your budget? So roughly this is, we just showed, we modeled how you can, how many individuals you would be able to identify if you use those specific cutoffs of LDL cholesterol. But it is an ideal, right? I mean, it's still missing 60%. So on its own, using LDL cholesterol actually is a, it's not giving good results. It's still a poor predictor for effect mutation. So then we together with Jasmine as well. She applied a machine learning algorithm on the UK, excuse me, the UK Biobank data to identify if there are other predictors that perform better. So for example, including not only the genetic but also environmental measures that could together improve the detection rate. And so this is a lasso model. It's a statistical method machine learning algorithm. And what we found was that actually triglycerides were one of the best predictors. The lower the triglyceride level, the higher the chance that an individual carries an FH mutation. I mean, it's not on its own, but in combination with other factors. So LDL cholesterol, of course, information on statin use. So relatively obvious things. We also included polygenic risk score. And we'll talk about that a bit more later. But here is the AUC statistics and you can see the LDL on its own is in blue. It's performing much poorer than the red line. And this is the lasso model. The lasso model definitely improves the prediction of FH mutation. And so if one has access to large data sets, phenotypes, genetic data, applying a machine learning algorithm can improve the detection of FH mutations using electronic health records. Perhaps we're not there yet. This requires, of course, some infrastructure as well. But I think this will be way forward in the future. Right. But I don't know if you remember now on one of my slides before I said, okay, but FH mutation can be found in about 20, 35% of individuals who are diagnosed with possible FH. So clearly there is a lot of people with high cholesterol who do not have a FH mutation. And this detection rate is much higher in definite FH cases of these individuals with tendon somtomas. But still, there is a percentage of sort of missing inheritance. So what are the reasons for that? I still, we can't explain high cholesterol, high LDL cholesterol in some individuals. And several issues probably could be highlighted here. And I will discuss them in the second part of the lecture. I kind of just want to leave you with those thoughts now for the break that will happen soon. So again, if you remember from some of my previous slides, majority of the FH cases would have a mutation LDL receptor, followed by the apple bead and about 2% of FH cases are due to PCSK9. And then we'll talk to you about next is the polygenic idea of polygenic hypercholesterolemia. And yet if we explain some chunk, I would say about at least 13% of definite, sorry, of possible FH cases having polygenic hypercholesterolemia, although it might be more than that. The proportion of individuals who are left without any explanation in terms of genetics. Some of the points highlighted that came out in our studies were that there were still technical reasons why some mutations were missed. Perhaps they are located in the gene regions that haven't been previously screened. So related to the previous old methodologies that were used, they were not data accurate, they were laborious, they were prone to human error. And I will show you examples of that. Perhaps really there are novel genes involved in the FH and I will talk to you about efforts to identify such novel cases. This will include whole exome sequencing, whole genome sequencing. Another issue is perhaps there is overdiagnosis of a FH. So if you remember the Simon Brum criteria, they are mainly based on cholesterol levels, family history. You would be surprised how many individuals that when I analyze the data for don't have a clear family history. And I think that's straight away a rings to me about, okay, perhaps that isn't the autosomal dominant FH. So perhaps they have high triglyceride levels. Those things, I think the overall bottom line from that is that the current diagnostic criteria in terms of clinical FH are not very precise. And I hope I just showed you as well that LDL cholesterol isn't a great predictor for an FH mutation. And of course the polygenic hyperchrystallemia, this is a relatively new idea polygenic risk course or the hot topic in currently in clinical genetics. How can we use them? Are they really useful? It still remains a question, but I will show you after the break exactly how, where we are with it and where we are going in terms of polygenic causes. Okay, so I actually finished my first part of the talk. If you would have any questions for now, that would be great. If not, we can break now and then come back after the break. So it's up to you now. I'm here to answer any questions. I really hope that isn't too much of a specific science. I think my bottom line is that I want you to understand that this is a good example of disease and it can be translated to many other genetic diseases. FH is great because it's relatively common and so we have loads of data, loads of resources to study it. And I remember when I was studying my PhD, I thought that lots has been done on FH and I probably won't have much to add, but actually the boom in next generation sequencing has enabled us to add much more knowledge to that disease. Okay, so what shall we do now? Can I speak to the organisers? Thank you, Martha, for such a nice talk and very informative topic, which is very common in I think worldwide. I have one question. If somebody visits the lab and he has the result that he has some high levels of cholesterol while doing lipid profile, particularly LDL, and he doesn't know about his genetics. So what is your suggestion or recommendation that he should revisit the lab and should do the lipid profile at how long time interval? I mean, if somebody tested today and he has high lipid levels and is there any recommendation from your group or internationally that should do it after a certain time? The DNA testing? Not DNA testing, lipid profile. Repeat lipid profile, is that what you mean? Yeah, so he can do the best for his lifestyle and he can manage the lipid profile in the normal way. I'm not aware of specific guidelines on that, to be honest. When you're saying that they are not aware of the DNA cause, do you mean that they would receive negative tests or that wasn't done yet? Yeah, because I'm just talking particularly related to our country because we are not very much facilitated with DNA testing. So most of us might have not been tested for lipid profile, with higher lipid profiles and not tested for these EpoB and LDL and PCSK9. So we don't know our genetics. But because you know this lipid profile is cheaper. So one can regularly monitor his or her lipid profile and then can do the best things which are suitable for the lifestyle. So I think the recommendation, yeah. I think the issue, so I'm not a doctor, but I know that the clinicians that I talk to, if they see cholesterol level, especially at the level of clinical diagnosis for FH, so let's say the 4.9 millimole per litre, that is high. So I know that they will prescribe statin straight away. They will prescribe statin and they will monitor the response to that statin. So the patient, I think if it's a new patient, it's kind of, it's still, the guidance are not specifically there, but I would say if it's a new patient that they would be seen probably in about six months time to see how they are doing on that statin, if they responding well, or if they need the dose to be changed, or even statin itself to be changed. So the lipid profile would be repeated at six months. If a patient is responding well, they are managed well. They would be seen in the general practice, I would say on annual basis. I know that FH patients, if they have a mutation, they are also seen at annual basis if they are responding well to their treatment, but they are seen by a specific clinic, a lipid clinic, so a lipidologist. So I think if cholesterol level is high, there is no need to wait. Triglyceride levels now, if these are high, they suggest that perhaps dietary intervention may work, but still as far as I know, statins would be prescribed. There is a clear evidence that the earlier you start statins, the better the projection is. So yeah, that shouldn't be delayed in a way. Does that answer your question? Does that help in any way? Don't feel bad about the fact that you don't have DNA testing set up yet. DNA testing for FH in the UK has only been approved about a year ago by the National Health System. Whatever we were doing before was research, so there was loads of sort of research. The charity, the British Heart Foundation Major Charity, was sponsoring most of the work before, so it wasn't very systematic. Yes, so yes, I understand that focusing on LDL cholesterol and lipid profile is a big part of it. Hello Marta, can you listen to me? Yes. First of all, I must say that it was a very nice presentation and you elucidated very well. Now coming to the question. My first question is that what I perceive from your presentation is that there is a direct relationship between FH and LDL, which is a bad cholesterol, whereas VLDL also come under the embankment of bad cholesterol. So what I want to know from you is that any relation between FH and VLDL and the second question is that you have mentioned multiple testing procedures for FH disease, but I was waiting that might be you will told us some preventive measure or some treatment regarding this disease because I have read somewhere that some herbs are there which can be very fruitful or useful for the lowering of LDL. So what's your take on it? Thank you. So VLDL we're not really looking into that to be honest. LDL is definitely more specific to FH. So I can't really tell you in terms of FH how to analyze VLDL. I don't think that's not the risk factor that we are focusing on. And as far as I know, in terms of cardiovascular disease, it hasn't got much value in terms of prediction and being a good risk factor. Your second question, so again, I will be talking about treatment on the next slide on the second half of my talk, but I am not touching it on any in terms of herbal or dietary interventions. This isn't recommended in terms of FH. FH is a very clear that it will lead to cardiovascular disease even if just so I mentioned previously that 20% of our cholesterol in the body comes from diet. And here we have a mechanism in terms of a FH is the sort of our body cholesterol production that is and clearance is not going well. So we focusing on diet is not very effective. I mean diet, you mentioned herbs. To be honest, I don't have knowledge on that. The only studies that I analyze is on statins or novel treatment that I will discuss to you, but these are not based on herbal findings. So, sorry, but that's beyond my expertise. Do you have studies that you think are showing good effect in terms of those? Yes. Have you ever heard about Oats Barley? Definitely you have heard about Oats and Barley. Yes. Myself porridge every day. So yes, there are definitely reasons. There is a long-term early peptide there as beta glucose. They are commonly found in Oats and Barley and they are very effective in terms of reducing the level of LDL and VLDL. Even they sometime it has been also observed that they enhance the level of SDL as well. So have you ever go through this? Okay. I apologize, I don't know much about it, but the recommendation is, so for example, in terms of FH patients, you need to lower the cholesterol level by 50%. So if you can do that with your... Marta, I will share some data with you. Okay, yes, you do. If the cholesterol can be lowered by 50%, I think that's great. And perhaps we will talk about the polygenic hyperperesterolemia next. And this is a disease where the environmental factors will have bigger play than the genetic ones. So if we move away, start moving away from monogenic causes where you really can't do much, but try to have sort of aggressive even drug treatment. In terms of polygenic hypercholesterolemia, the dietary or your lifestyle has much bigger effect. And perhaps agents, as you just mentioned, may have maybe more successful. Okay, thank you. Okay. I think there is a question. You're welcome. I'm just getting, thank you on the chat. So shall we break now for... We have one more question, Dr. Marta. Say that again, sorry. Hello, ma'am. Hello. Good afternoon. I'm Dr. Amina. I want to ask one question. What is the identification criteria for FH patients? Are your universities linked to any tertiary care hospital or anything else? So identification criteria, as I mentioned to the clinical diagnostic algorithm or the criteria, the Simon broom, the Dutch scarring lipid system. So I think majority of FH cases would come from GPs, the general practice. And I mentioned in England, individuals at the age of 40 are offered health check, which includes cholesterol measurements. Today, their cholesterol is above those cutouts for clinical diagnosis of a FH. They would be then referred to lipid clinic. In terms of hospitals, I think these are the cases that would come with a heart attack, microinfection. So already a cardiovascular event. And that's how I presented to you the Mike's family. So it is in a way too late because the disease wasn't prevented, but they would then come that way. So in terms of identifying systematically, the only implemented at the time system is the cascades testing. So once you have an index patient, you would be screening their family members for a specific mutation. And the child parent screening strategy is currently being tested and hopefully will be implemented too. So these are the kind of efforts that we have at the moment in terms of identifying the individuals. Thank you so much, Pam. Welcome. Okay, shall we have that break or otherwise we'll miss and I really need my drink. Sorry, my throat is going dry. I will just pause the sharing. Hi Martha. Now I can see you. Hello. There is another question. Okay. Good afternoon, Martha. How are you? Hello, I'm good. Thank you. I'd like to have a question. You just answered Dr. Amna's question that how the patients reach the samples of the patients that reach your lab. But in a developing country like Pakistan where the research centers are absolutely, you know, out of touch from the tertiary care centers or the hospitals. How can we do screening or analysis? Because I'm pretty sure this particular familial condition is very much prevalent in a country like ours where consanguinity, the culturally promoted. Yes. Culturally and socially, most of the familial disorders are linked to intermarriages and consanguinity, dozen marriages. So it's very much prevalent in Pakistan and probably we have a very good pool for this particular disease. I'm pretty sure. I'm more than sure because the South Asian population, India, Pakistan, Bangladesh has the same cultural norms. Well, that's another thing. So what strategies would you suggest in which we can collect the sample and bring them to the labs? It's a very challenging for the very challenging who are sitting in the labs we have we wanted to work, but we do not have avenues where we get the patients sample into the labs. Yeah, I see. I'm just thinking so I'm in touch on LinkedIn and Twitter with a person. I'm going to bring his name after the break. As far as I know he's been putting some efforts in terms of identifying individuals that just very recently started in terms of children in Pakistan. So, I will try to find the contact. So this issue is big and I understand it's a problem and looking at the experience of UK where we have the resources in a way you would think we can do it much quicker. It has been going very slowly still and not only UK but Europe I think the Netherlands was doing great job. Sorry to interrupt you. Then again we've got conventional methodologies for excluding the genetic variants like you know we've got reflip analysis or just small regions which we analyze. But we have meager funds for next generation sequencing or things like that, unless we have a grant from some Institute. So these are things which are really restricting our energies and I would say capacity of doing work. Yeah. Yes, it's unfortunately still not quite there isn't it in terms of, we have the knowledge but to implement it in terms of preventing cardiovascular disease it's a, it's another story. I understand it will involve not only scientists not only clinicians but the public health specialist and then it's a big logistical operation to implement such thing. I suggest. I mean, the only thing, every country is specific that's the thing so you will know exactly what your needs are I mean we need to sit down with others to work out what would be the best place. You could learn from other countries. We have started doing such things so the European atherosclerosis society meeting is coming up in May. And as far as I know so there is a initiative to to drive the diagnosis of FH so there was a big Lancet paper recently of different countries overview and how how things can be moved forward really you know FH is under diagnosed and we've been talking about it for years now and not much has been done. So yeah it's a I understand your your in a way frustration. And I just really hope that something can be moved forward. As I said I will try to put you in touch with us and you did the content detail of the person who is trying to do something. Yeah, but I understand it's it's not an easy task. Thank you Martha. You're welcome. So can I, is there more questions now. No matter, I think it's and do you have another session or. Yes, so I do have more slides after that we will cover what are the reasons for the missing heritability in FH we will cover some novel treatment and how you go from genetics novel treatment. Yes, there is still more more things to cover if that's okay so shall we have a have a break. What was the plan break now was it 20 minutes or. It was, it was but now you can continue if. Oh I see. I need five minutes if that's okay. Okay. I need to get myself drink my throat is gone very dry so. If you give me five minutes I'll be back. Okay. Thank you. Even fight. Hello everyone. Everyone kind of back or should I give you more minutes. Welcome back. You can continue now. Okay. So just want to so I want to share with you my screen. To show you the person that I mentioned. Yes, we can see now. So motors also bar. I think he's from Iraq but he's been hosting the news about Pakistan. And so I mentioned that there is some sort of effort started quite recently so this is a month ago. When he mentions his colleagues being Khan the director of newly established Islam about the electric liquid clinic in Pakistan diagnosed the first to homozygous FH cases. Based on the FH symposium delivered. To them so when I think. Yes, so this is exciting news. I don't know if you are aware of. Of those cohorts of those efforts to identify FH cases. This is children so I think that's that's that's amazing but there is clearly something already starting so you might want to. Try to get in touch with this person. Or with colleagues being Khan. And as I said Islam about pediatric liquid clinic in Pakistan already diagnosing children is wonderful news. So if yes I'm not exactly sure what the national efforts would be. But that person will be definitely more in place to help. Did you take a note of that because I want to now swap. I'm going to go back to my slides. So we were here I got my coffee. Just before the break I mentioned to you that we were still lacking in terms of identifying genetic causes of FH. Only about 20 30% of possible FH cases which is really the majority of the page patients have a mutation. So in a way you might say well the genetic testing is kind of it's not very cost effective right if you only confirm the diagnosis in that proportion of patients. Still very important to do I think if you have the resources. And as a colleague in the audience suggested that perhaps in Pakistan. You might have quite few or a higher percentage of homo zygote cases I think it's a very important to know the mutation. So let's let's think about it what are the reasons for the missing FH mutations and one of the first one that came up was technical reasons and. We learned about it because there were significant advances in sequencing technologies from the previous methodologies. In our study thing this is back in 2012, where we applied whole exome sequencing, we identified 25 FH mutations out of 125 patients who should be. The mutations should be detected using the previous methods such as the HRN. So that was quite shocking it's a quite big proportion of patients that we we said today. They are mutation negative but they actually had a variant is just the technology perhaps wasn't quite there. Another issue is that we are still not really looking into the deep intronic spaces in LDL receptor, we're only focusing on the coding regions. This is where we can easily interpret we're not easily but we can start interpreting the mutations because they affect amino acid. So we kind of can predict what's what's going to happen when you have a mutation in the coding region. But obviously the intronic regions are actually much larger than the exomes and they might affect the correct splicing of the gene. And there was a study, for example, one of the studies back in 2018 showing the deep intronic region sequence they they sequence the whole sort of intron here and specifically between X and 14 and 15 of the gene. They identified there was a cryptic splice site which meant that when the gene gets translated into a protein, there was a retention of region of a DNA which shouldn't be there and that obviously caused an issue. It shifted to the whole reading frame of the of the gene and there was a then premature stop code information just in the X and 15. So these variants, you can see it's it's quite deep into the intron. Why were we not looking. They do affect small proportion of individuals the estimated point 24% of all FH mutation negative patients, but still think are if we want to pinpoint the missing causes. This is important part of DNA that we are not looking at and we were not looking into those regions because of they are very rich in repetitive sequences specifically out of sequences, and they are very difficult to sequence, technically. So, yeah, it's one again reason why perhaps we can't find a mutation. I want to talk about the over diagnosis and maybe our discussion here was kind of leading to that. Clinically the clinical diagnosis of a feature mainly based on LDL cholesterol, but of course you if you see a patient, you will see loads of individuals with high cholesterol. Now the question is how high. What are the other cholesterol fractions like so for example triglycerides triglycerides are the indicator of in a way dietary cholesterol. And when you look at typical FH patient they are actually quite lean, they wouldn't be having much issues many issues with diet. So, their triglyceride levels are actually low. And I did the study on this is a sample for about 300 individuals from a clinic around Oxford area in England. And if you take. So we were looking at the mutation detection and how we can improve sort of diagnosing of individual who actually have a mutation. And I'm, please don't take me for this I'm not saying that those individuals who have high cholesterol but don't have a mutation or not important or not be treated. But as I presented to you before is that having a mutation is an additional risk factor that's why I'm focusing on those individuals. So additional risk factor to plus the LDL cholesterol, that's what it leads to very high risk of premature heart disease. Nevertheless, all patients who have high cholesterol should be treated. So you can what we showed in that study, we achieved 100% mutation detection rate in individuals who had total cholesterol in this case total cholesterol levels. The top quartile and triglyceride level the other side on the bottom quartile. So if you have very low triglyceride level and a very high total cholesterol, we were able to achieve 100% mutation detection rate in those individuals. But there is a suggestion that perhaps taking the triglyceride levels into consideration when clinically defining a fate will improve diagnosis or detection rate of a patient patients. And of course the family history I said that now probably three times but looking into that pattern of 50% first degree relatives being affected in autism or dominant manner is again strong indicator of that individual carries an imitation. So yes, there is a probably still issue of the over diagnosis or that our clinical diagnosis is not quite accurate. So let's have a look now into the polygenic cause and this is a cause that majority of your patients will probably have or this is majority of those who have high cholesterol level that you might see in your clinic. So how did we come about defining that something has a polygenic cause. And this was enabled because of the genome wide association studies for lipids. And back in 2010, there was then the largest study of over 100,000 individuals where they had genotyped SNPs. So here we're talking about SNPs. These are single nucleotide polymorphisms, not mutations. So I always want to separate those two. Mutation is a rare variant that has a strong effect on the phenotype, in this case LDL cholesterol. So on its own can cause disease, whereas you've got SNPs, these are polymorphisms. So you and me will have millions of SNPs in our DNA. And that's why we differ. And these variants are common. They are, you know, in one population, it might be more common than in another. But they're relatively common. And on their own, they will never lead to a disease. If you have one SNP here that affects LDL cholesterol. It doesn't have a much of an effect. But if you imagine taking them together, they might have an effect. So that's what we wanted to test. How many SNPs affect LDL cholesterol? And what happens if you inherit more than average of those SNPs? So we did take those polymorphisms from that GWAS study. Now, bear in mind, this is actually quite old now. This is a recent GWAS study, also from the genetic, the Global Lipids Genetics Consortium. There's a nature paper that just came out at the end of last year. But back then in 2010, there was 95 SNPs significantly associated with cholesterol. And each SNP had a modest effect, right? This is the beta effect. So that's when you do the statistics on the GWAS study, you will get beta effect. And that shows you how much a specific variant modifies the LDL cholesterol in this case. So in combination, the total set of those variants explained a considerable part of the variants in LDL cholesterol, and that was 12%. So we can use those SNPs in combination to calculate polygenic risk score. And that's how polygenic risk scores. That's what they are really. So what happens is you genotype an individual. So we've got an individual here for several different SNPs. In this case, I'm only showing you an example on two SNPs. So we've got SNP1 and SNP2. From the GWAS study, we know that the A allele in SNP1 is associated with higher LDL cholesterol. So we genotype it. We've got a homozygote for the A allele. And then we've got a heterozygote, another individual heterozygote for the A allele and then C allele. And then there is a homozygote, another individual for the alternate allele. And we do this for a number of SNPs. So SNP number two in this case is the T allele that is associated with higher LDL cholesterol. And these were the genotypes for the three individuals we looked at. So if you then want to translate into numbers, you would say the LA allele is the lipid rising SNP. So if a person has got is a homozygote for that SNP, they will get two points. So one for one A, one for another A. If you are heterozygote, you'll get one point for one A. And if you are homozygote for the different allele that doesn't raise LDL cholesterol, then you get zero points. And you do the same for SNP2, where the T is the risk allele as we say it. So if you are heterozygote, you get one. This person gets one point as well. And this one hasn't got any T's, so they get zero. So based on those two SNPs, we can calculate a score. This is very simplified, but you can basically add the number of risk alleles. So for individual one, we add two plus one is three. That's their gene score. And so on. But that very simple calculation doesn't assume or assumes really that every SNP has the same effect, which isn't true. So from the dual studies, we know that every SNP has a slightly different effect on the LDL cholesterol. So then we generated a weighted gene score where you multiply the number of risk alleles. So from that simplified score, you can then multiply them by the specific beta effect that the dual studies, the statistics analysis provides. So if you multiply by that, you will get a slightly different number. You again sum up the number and you end up with the weighted score. So that's the simple idea of polygenic risk scores. I've been saying gene score, SNP score, so you can see that there's a different vocabulary here, but it really means the same thing. I think these days people don't want to refer to polygenic risk or if the score is based on the on 20 SNPs. If you look at all the nature papers, they these days use millions of SNPs and that's when they kind of reserve that vocabulary of polygenic risk score to when you have 100,000 of millions of SNPs together to generate one polygenic risk score. So I will be saying either SNP score or gene score in this case, but it really means the same thing is just a matter of numbers. So we from that Geo study, the original Geo study, we took SNPs that are that have the biggest effect, so the beta effect on LDL cholesterol and SNPs that are the most common, and we identified 12 SNPs. We then calculated the gene score based on those 12 SNPs as showed on the previous slide, and then we plotted that score. Now this one is for the not weighted one. This is just the number of risk alleles, and you can see that's the distribution in the cohort of Whitehall 2 population. So this is a kind of a cohort of civil servants from England who donated their DNA, who donated their lipid levels, just over 3,000 individuals, we managed to do the score. And what we've shown was that the score strongly correlates with the cholesterol level. So here's the deciles of the gene score, so the higher the score, the higher the LDL cholesterol. So it isn't binary thing. It's not like having a mutation and not mutation. This is continuous. It's almost like the distribution of LDL cholesterol right in a population, and it nicely correlates with the LDL cholesterol. I want you to bear this in mind. It explains only about 10% variance. That means it's not, again, very diagnostic. But it is a tool that shows you that those individuals who are in a top decile of the score have much higher, significantly higher cholesterol levels than those in the lower deciles. So clearly there is some association. And we call this a genetic instrument. So this gene score was kind of a genetic instrument that allowed us to look into individuals with a ph. So this is all done on the sort of healthy normal, let's say population. When I say healthy, obviously not many individuals are healthy in the general population, but just an average sample. So we had that control sample from the White Hawk to cohort. And what we then decided to do is to genotype for the same gene score, individuals with a ph who don't have a mutation. So they have a clinical phenotype of a ph, but we couldn't find any mutation in their genome using the, that was I think the targeted sequencing method. And what we found was that individuals with a ph, but no mutation had significantly higher gene score in comparison to the control. Again, you can see very big overlap. But there are some individuals who are really at the extreme of the gene score. And that really suggests that there is a polygenic component plays a significant role in terms of individuals with the clinical ph, but no mutation. And so this can information can be also used in terms of identifying novel variants for a ph. We think those individuals who have very high polygenic risk or we can kind of explain them and say your cholesterol is due to those steps. You probably don't have a mutation otherwise you if you have mutation and polygenic risk or high, then your phenotype will be very extreme. We can also use that information and say, have a look at those individuals here. They've got very low gene score, even lower than the average healthy population. But they don't have a mutation identified so perhaps whole genome sequencing searching for novel genes in those individuals can be a good idea. And another conclusion from this was that testing a cascade testing so when you trace within family individuals in the polygenic hypercrystalline will not be that effective because the inheritance pattern of polygenic cause is different than monogenic monogenic. It's clear you've got 50% chance of inheriting your mutation from affected parent, whereas with polygenic when you've got in this case 12 SNPs inheriting them they are all independently inherited. Inheriting 50% of those, the chances much lower. So we think that about 30% of polygenic FH cases would be having, would be passing on that disease into their first degree relatives. And on those bases. That's why it isn't cost effective. So compared compared to the white hole to a population the control in the FH mutation negative group 54% had a score in the top three decile in comparison to only sorry, in the top three decile. And only 11% in the lowest three decile so very different from the control normal distribution. We then replicated those findings of the gene score in feather cohorts, including children. In this study we had a Dutch children Netherlands and Greece and they actually had the highest polygenic risk and I think this is interesting because that's so just if you take children population the effect of environmental factors is not as pronounced as in adults. And so they are kind of more pure cohort and it makes sense that they have the highest genetic reason polygenic reason for their hyper crystalline. They don't have mutation but they have very high polygenic risk score, and they do have the funeral time. So those studies on polygenic score in in familial hyper crystallinia have provided some evidence again for the for the health policy for the nice guidelines. Since then the cascade testing resources are really recommended only for the monogenic for the mutation carriers based really on the cost effectiveness of it. And another guideline for research future research is to focus on those who have very low polygenic risk score and don't have a mutation so they are more likely to carry a novel gene. So again it's in a way an example of precision medicine here how the different genotype has a consequence as well on the patient management. So patients with monogenic we reserve much more resources on the monogenic cases and I will show you on the diagram later. These guys they get more attention in terms of management in the lipid clinics by specialized lipidologists, whereas individuals who have polygenic cause and they are more likely as well to respond to treatment, much better easier. They could be managed by general practitioners, which is cheaper way. So let's have a look how you can go on patient stratification in the genotype so if you have a patient, an FH patient comes to clinic. I mean, so you don't know at that point that they are FH but they have LDL cholesterol 4.9 millimole per liter. They have a strong family history and they have tendons and tomas. So in the UK what we would do. You would send that sample for the next generation sequencing test. And if the mutation is detected in one of those known genes, then that patient is referred to an FH nurse. So we've got now FH nurses who are very good at performing the cascade testing. So they would contact all first degree relatives. They have from this test the information of the mutation and they would send the DNA of the relatives for confirmation if they inherited the cause of FH. But in most cases, the FH patient will not have a mutation detected. And so what happens then. We would determine the 12 SNP gene score. And that is actually included not officially but it is included in that DNA test. And if they have a high gene score and there is a high probability of them having a polygenic cause. We do not cascade test those individuals. Because there is a lower detection of affected individuals. Remember 50% here chance here probably around 30% chance. So it is not recommended someone has calculated the cost and said this is not cost effective. If that's a right choice or not. It's not for me to judge here but that's that's how the guidelines are at the moment. So how about if an individual hasn't got the high gene score but they've got low gene score. So now you're thinking oh gosh they haven't got a monogenic cause they haven't got a polygenic cause. So you could refer those individuals for research to look for other novel causes of FH. And this is where the 100,000 genomes project in the UK comes in and I will talk about it a little bit more later. But it is really looking for novel causes in that population is likely to be more affected. But the bottom line is treat the phenotype and you cancel the genotype. So still goal is to lower the cholesterol level of those individuals. No matter if they're monogenic or polygenic. So what could be the novel causes of FH then? And why do I bother about it? I will tell you after showing you some of the studies how you can go from identifying novel gene to a novel exciting treatment. So our first goal at looking for novel causes of FH was being involved in the UK 10K study by using whole exome sequencing. We had the chance to sequence 125 definite FH cases. And that is when we found out that 25 of those individuals had actually mutation in the known genes. And that gave us some lesson there that our current methods were not performing very well. We then analyzed the genetic score, the gene score in those individuals. And further 29 patients had a high polygenic score. So we removed those from the further analysis. We then ended up with loads of variants still. You can imagine whole exome sequencing generates loads of data. So we had to filter all those down. We ended up comparing all genetic variants in 71 unexplained FH cases versus almost 2,000 controls who are sequenced as part of that project. And we identified some interesting genes. The top genes were CH25H and INSIC2. And to tell you that the statistical suggestion is still that those, the significance is not quite high yet to be certain that those genes are causal of FH. So it is still work in progress. And unfortunately those sequencing 125 individuals, it's not enough if you want to identify novel cause. So we need to enhance the power of the detection by building bigger cohort, probably more diverse as well. So yes, we then moved to the 100,000 genomes project. So 10 times bigger. And this is genomes now. So we're not looking into only the coding regions, but here into the whole genome. So all the intronic spaces, all those untranslated regions. And this is part of a big initiative that was paid partially by the government in the UK, where we invested into genomics research. Many startup companies since then appeared on the market in the UK. Lots of people were doing whole genome, whole exome sequencing, etc. So it was a big change back then. We do have a cohort of FH patients again in that project over almost 500 really I think now individuals. And they are part of the cardiovascular disease domain so there are several other cardiovascular diseases being sequenced at the same time. And we had an initial look at the data in the FH cohort and unfortunately again we found some known mutations. So bear in mind those individuals that were referred to genomics England should have previously had the DNA testing for FH mutations in known genes. But for some reason, some of them were missed. And so in total, we explained 15% of those submitted cases as to who had a mutation in the known FH gene. This is first time we also managed to look at the apple E mutation that was quite recent. And it was interesting to see that there were 10 of those individuals had the apple E mutation in brackets you've got a number of different variants. So obviously mutations in LDLR was common. And so there were 54 cases due to 38 different mutations. Having the whole genome sequencing we also looked at the 12 SNP score. And if you take the 75th percentile cutoff. We identified polygenic calls in 29% of the recruited patients.  So the next so we would assume that the monogenic in known genes are explained. Those polygenic 29% cases again are explained so we would focus really on those with lower gene score no mutation to do a burden test analysis. So here we've got being part of the big project we have a chance of comparing variants in our FH cohort to those other cohorts within the project who are all sequenced in the same way. So at the moment where I was looking at is that she was hits. So these are good candidates for novel cause of FH. So these are SNP genes that were shown to be associated with LDL cholesterol in the large dual study. So as I said there was a recent large dual study published end of last year. So I'm currently running those analysis. We have looked at there is a recent paper suggesting RNA processing being playing a role in terms of LDL receptor gene metabolism. And again I'm looking into those specific genes and I'm looking at any other gene in the genome. But I think again the bottom line is that novel cause of FH is likely to be very rare and possibly unique to a specific mutated family. And so the success of such studies really depends on the sample size the bigger the better and the more diverse the better. Although novel gene will not significantly improve the diagnosis yield because we will not increase the percentage of detection rate by drastic measure. But it might lead to a new drug target for LDL lowering. And that leads me really to the next slide. Actually now here is sorry just a summary as well what I've mentioned to you about the RNA processing. So you might want to look at that paper it's a very a lot of science to be honest packed into this one report. We did that in collaboration with colleagues from the University of Zurich. So another method of identifying novel causes of FH it's the approach here was to do genome wide RNA interference screen. So this is for genes that limits the uptake of LDL cholesterol in cells. And what happened with the results shown that 54 genes were significantly lower. So maybe so maybe I'm going to fast but this RNA interference screens means that you knocking down every single gene in the cell and see if that knockdown will have an effect on the uptake of LDL cholesterol. And indeed 54 genes. When you knock them down, they had a significant impact on the LDL cholesterol uptake or clearance if you like from plasma. And 15 of those 54 genes and code for components of you to splice a song so this is kind of like a machinery that controls correct splicing of genes. Knocking down any one of 11 of those 15 genes regularly resulted in a selective retention of interim in LDL receptor. So clearly something went wrong if you knock down some of the splice of some genes. And then the whole machinery was not able to correctly splice LDL receptor. And so there was a retention of gene regions that shouldn't be translated. This is in a way the junk DNA that should not be there should be spliced out cut out. But it. Yeah, it led to that and that had effect on the LDL receptor function. Now, RBM 25 was one of those genes, and that gene actually came up associated as well with FH phenotype in my UK 10 case study so the whole exon sequencing study. So this was quite exciting is that's another sort of evidence from having those initial genetic findings now to this sort of functional study, showing that perhaps that gene is important. Again, more studies are needed to confirm that. Because it's very rare we only have three patients with that with variants in that gene and saying that those variants are actually truly pathogenic it's another challenge. So, from those rare novel findings how can we go through to effective treatment, and I will get to that point but first to talk about treatment we need to cover what is the current treatment available. And initially, it was the fat restricted diet that we had perhaps a little bit touched on during the discussion. But that's reduced cholesterol only by about 10 to 15%. So that wasn't enough for an FH patient. The first drugs license where cholesterol binding reasons, which were really not correlated well and again the cholesterol reduction wasn't very effective. In 1990s, the powerful and probably the most prescribed drug statins was discovered by Japanese biochemists. And the first statins would lower cholesterol from 25 to 55%. So that was a big, big difference from the previous efforts. And although studies we do know that they have some side effects and some patients do not tolerate them. They do have significant impact on the survival. And this is on FH patients. Cumulative event free survival. When we're talking about event is the CHD event. The first statin treatment is the red line and the blue is on statins. And the difference is it's it's clear and I think if you have an FH patient they need to be on statin. They will benefit from that. So let's think about how statins actually work. I hope you would recognize this if you're not a scientist and maybe not biochemist. So this is the, sorry my, not quite working. This is the hepatic synthesis of cholesterol. And so we go from the beta oxidation of fatty acid through to several process until the cholesterol is actually synthesized. And that process is tightly regulated by rate limiting enzyme HMG CoA reductase. Obviously the cholesterol then goes to play important roles in the body. And statins are actually inhibiting, they are inhibitors of the HMG CoA reductase enzyme. So very slightly sort of cancel the function of that enzyme. And so this process of synthesis of cholesterol is reduced. If we knock down that enzyme, then the cholesterol synthesis is lowered. And so what happens if your cell, if your liver doesn't always unable to synthesize your cholesterol, it's cholesterol because it's enzyme is inhibited. Then the liver will try to express more LDL receptors so that the LDL receptors can pick up cholesterol from the circulation. Okay, so that's how it works. That's how the cholesterol is lowered in the circulation in the blood. So it clears the cholesterol from blood because it needs it for its own purposes, because its internal or hepatic synthesis of the cholesterol has been inhibited. So it really works through upregulating the LDL receptors. Anyway, you remember from the genetic studies. Now, if you remember from my first slides, it is the LDL receptor that needs to be functional in order to clear the cholesterol. So here's a little catch.  So you don't have anything to upregulate in a way. But the statins on homozygot cases do not work very well or at all. And that's why homozygot cases needs a completely different treatment, which I will cover. But yeah, I think we need to bear that in mind that hydrozygot cases have 50% so that they've got one copy of a gene that is working well and one copy that is faulty. So we can only upregulate the good copy of the gene here. But again, you can't go completely normal in a way if you still have one 40 copy of LDL receptor. So now I just mentioned to you that homozygot cases, the heart disease in those children would be evident by about five to 10 years of age, which is extremely early. So instead of in a diverse population, you've got frequency of 1 in 300,000 individuals, but perhaps in Pakistan that could be higher as suggested. And poor response to statin there is no normal LDL receptors to regulate. The idea to treat those cases, if they're not responding well to the statins is to introduce LDL aphorases to extend life and LDL aphorases. It's kind of like dialysis of blood, where a patient lies down and they've got the tubes connected. So that's where their blood comes out to this machine where the allele cholesterol is cleaned, just like dialysis really and then the clearer blood comes back. But you can imagine this being a very expensive. The patient needs to travel to the center. They're actually from about seven years ago, so they've probably everything has gone up in price. So I suggest this. This is underestimated. Patient need to travel needs to be. This needs to happen in about every two weeks, or it to be effective. So it's tragic for those homozygot patients, they really depend on that treatment. They require a liver transplant. You wouldn't want to if you want to know a little bit more about homozygotophage is this very recent paper. The experience of homozygotophage retrospective cohort study, a large study that was just came out in the Lancet in February so some of you might be interested in finding out, but that is the story for homozygotophage at the moment. So that of course then drives people to identify new treatments for ph. And one of the ideas was a drag called mypermersone, or kinamerum, which is a short 20 base per single stranded anti-oligo that should bind to the apoB mRNA. So we in a way knocking down apoB. Now, is it a good idea if you think about it. So this would affect the synthesis of LDL in the liver. So the liver obviously can synthesize its own cholesterol and then let's out to the circulation. And when it's in the circulation, it is wrapped around with the apoB. So the idea here was that if you knock down the apoB, the cholesterol will not be synthesized. There will not be exiting the cell into the circulation and increasing the cholesterol. It has been approved by FDA for use in treating homozygotophage, but it has been rejected twice by the European Medicines Agency. And the reason for that is that it is associated with fatty, so here secretion of VLDL and fatty liver. So you can imagine that if you knock down the apoB, think the cell, it does, it still synthesizes its cholesterol, but the cholesterol cannot exit the cell without the apoB that is knocked down. So it starts accumulating in the liver. Okay, so it isn't a great idea, but it is an option in the US for homozygot cases. There are better drugs on the market that can do a great actually LDL lowering. One of those is with two of the drugs actually, so there are two different companies develop drugs almost simultaneously. They are fully human monoclonal antibodies against the PCSK9. So PCSK9, if you remember from my initial slides. It's an enzyme that regulates, let's keep it simple, the great LDL receptors. So if you knock it down, you will not have the degradation of LDL receptors and there will be basically more receptors on the cell surface available to bind LDL cholesterol. So that's the idea behind PCSK9. It was based on the discovery of PCSK9 mutations in FH patients, gain of function mutations, if you remember, and also on the fact that in a general population, if you do a GWAS study, for example, PCSK9 always comes up as lowering LDL cholesterol. Confusing you, I think, but loss of function variants in that gene associate with low cholesterol level, whereas gain of function associate with FH phenotype. So in one gene, you can have two different types of variants that lead to the completely opposite phenotype. And that in a way gives us a good control, genetic, natural control. And there are two reports of apparently healthy individuals with no circulating PCSK9 as they've got a mutation that removes that gene.  And that in a way suggests that this is a great control for that drug to go ahead. Because if you knock down in PCSK9, as in those two individuals reported, then that will not have any side effects. And indeed no major side effects using PCSK9, which are both approved since 2015 have been reported. And PCSK9 is, it's a great target. And more companies, drug developing companies have picked up on that target. And here we've got a synthetic small interfering RNA made by Novartis. They came up with a drug again to inhibit PCSK9. So the same mechanism, slightly different delivery twice a year. What they're doing is they having an injection, they call it even a jab. Twice a year, a person would come to the clinic to have an injection with that small interfering RNA agent, which would lower their cholesterol for the next six months. And in the UK this made news, it has been approved in September 2021. Somehow the nice, the policymakers made a good deal with the drug company. I'm not involved and don't know what the deal was because the drug is still quite expensive. But it's there if it's needed. I think if there is, of course, specific guidance, who can use the drugs, but it is available. So another super exciting thing is a treatment based on gene editing. And I believe Ali Aqsa has presented to you something on gene editing. So this is kind of similar philosophy in a way. To modify a gene. And if you do that obviously that will stay for for the lifetime. And that way you could have an effect on LDL cholesterol. There's a company called Ver Therapeutics based in the US, who have published already this in vivo CRISPR based editing of PCSK9. And that was done in monkeys in primates, and it lowered cholesterol beautifully in a way, and it lasted for a long time. And they still got those monkeys but basically it's a lifetime one off treatment. So what they're doing again they targeting the PCSK9 gene, they modifying a single base pair that will lead to a premature stop coden in the PCSK9. Yeah, it's basically knocking down PCSK9. The idea is to do it once in patients life through the liver. Obviously there are issues of in terms of off target effects. What if you injected would you really sure that it will modify only one base pair in a specific gene. They've got data showing that yes, that is the case. So very exciting. They're planning a clinical trial in human to start soon as far as I know. So watch the space data that might be a breakthrough soon. So that is where we heading and I just wanted you to understand that this is coming from the genetic findings in a disease. So from from knowing a very rare mutation in PCSK9 that accounts for about 2% of FH cases. So a rare variant that does not have bigger impact on patients on the mutation detection rate, but provided incredibly important information in terms of identifying novel drive. From the actual genetic finding of PCSK9 to development of the antibodies and having approved the drug for was about 10 years time, which is incredibly quick for novel drug development. So, yeah, genetics more and more has been proving to be aiding the way we develop drugs. And they the genetics really play important role now. If you want to test novel drugs. The PCSK9 is a great example. Okay guys so I've prepared for you a few quiz questions and I just want to for you obviously I'm not going to mark this but I want you to kind of try to reflect on what we've learned today. See if you can answer those those four questions. I guess, you can always go back to your slides later. This is very FH specific talk I realized that and I know this high cholesterol affects many more not only FH patients. So, yeah, again, I think it's a good model disease that you might be able to translate in the future to other things. So I'll give you five minutes and please think about those questions if you can answer them just a little exercise. And then we will have a look at the answers. That's okay. I won't be checking individually with you. Hi Marta. Hi. Thank you. Thank you for such a long session and management about this disease. And I'm looking at the participants. They have answers. They do. Yeah. Okay, excellent. So, I hope you remember that FH affects one in two hundred and fifteen individuals. So the most common Mendelian disease remember that there isn't any other ones that I know that is that common. The gain of function mutation. I think I said that so many times now and it has been evident throughout talking about the treatment. PCSK9 is the gene where gain of function mutation as opposed to loss of function mutations cause of FH. What is the most likely cause of FH in mutation negative patients? You could probably think for a while here but it is the polygenic cause. So the average person that you will see in a clinic as a doctor will have a polygenic cause, a combination of genes, common variants and a lifestyle. And start to introduce LDL cholesterol by increasing the expression of LDL receptor. So knocking down the important enzyme and synthesis of cholesterol would drive expression of LDL receptor. So I hope you got some of those answers right. I will just leave you this summary slide again. I think we probably summarized quite a bit in the quiz. But yeah, it is an inherited disease of high LDL cholesterol, very strong risk of CHD. Unfortunately, it remains underdiagnosed and under-treated. So even if you have diagnosed patients, still some of them are not receiving the treatment, appropriate treatment to lower their cholesterol. As I said, the European guidelines say lower LDL cholesterol by 50% from the baseline. Mutations in genes involved in LDL cholesterol pathway cause of FH, the frequency 1 in 250. Polygenic hypercholesterolemia mimics the monogenic phenotype. Knowing the genetic cause is very important in terms of the monogenic cause as it helps to have a cost-effective screening strategy. And genetic findings in FH, I hope I've just demonstrated that, can generate important knowledge for novel drug development. So last but not least, some of my acknowledgments here. Obviously I've presented many other people's work, hopefully cited them, but yeah, thank you very much for your attention. I'll stop sharing my screen, so thank you. And I'm sorry if I have been talking too much, but it's my favorite subject, so I can talk all day. Thank you so much, Dr. Marta, for joining us and spending time for us. I hope participants have learned some knowledge out of it. And thank you so much, participants. This brings to end of the third day of the session. So see you tomorrow, inshallah. Thank you so much. Thank you. Thank you. Thank you, Marta. Thank you so much. Thank you. Take care, everyone.


Welcome to BCH technologies this Kevin to the just a short video about the Epson Zero X 69 air and I got two of them this month a kind of weird air and I did a lot of research and Okay, one thing is on On the YouTube there is a fake Videos asking asking to download the software or there's a fake website as asking to download something or install something and don't go for it and Apple couple hours on the phone with Epson, okay? and Epson asked me to send that a printer back and they said they could go and give me a new printer and just as soon as I Hand up the phone with Epson and I get my unit solved So I cannot repeat the air. So I just show you how I solve it. It might not work for you But it worked for me today. Okay, so basically zero X 69 is something with us with a cartridge movement So we come seeing it has a block here to prevent it prevented the cartridge from moving movement, so you're supposed to remove this block and So what you have you have air here in red say something wrong and this zero six six nine and One way to either troubleshoot it is to power the printer off and So here's that the door trigger Or the lead trigger so if you stock a piece of paper towel in it And you turn up power back on The printer will think of this and this This door is closed. So it won't bother you with please close the door. So You can easily see the movement here First thing absent asked me specifically if I see anything blue here Blue packing tape is so there's none and that they asked me if anything around here and I don't see anything that's blocking the movement and So next thing absent is hey, do you want a new printer? I say, yeah, of course. I want a new printer So they're gonna send me a new printer Okay, so now we turn it back on The first error you're gonna see is a yellow error said this door is not latched. This is really weird So I can still cannot figure out the where's the sensor Frightened to tell if this door is not latched Because I already already resolved this error. So it doesn't have that so I turn it back on so if I still get a error you're gonna see a yellow window right right now says this door is not latched and It has you to click okay And after you're clicking the okay It's gonna start moving a lot a little bit So if you see here, what you want that you want that the thing moved to the middle and then you cut the power off Okay, you unplug it So what you can do is I you just you say okay with that yellow on that yellow warning and they think start moving You just like this Okay, so when you move the move to the middle you cut the power off Okay, I I let them move too far. So now it's locked on this side. Okay. It's okay as long as this whole unit move out of here and You can work on here. So here's a here's a little sucker and It's a we'll do the cleaning is suck sucked in part of it and here's the bladder So you just use your hand make sure everything is Make everything just to move a little bit and What I found what happened to my was This assembly right here, okay So this piece is protecting protecting it from movement During the shipment something some something in the shipment that knock this off the same Not this off the track or something. I don't know it's just you know Probably it's in the position and you can see So you can have two different positions. So just change a little bit Okay, I'll change it here and so this thing This thing is a it's a printer lock is supposed to pop up and the lock the printer so basically use your hand to play with this little bit change a little bit and then That's it. So now I power it on now Yeah, I'll zoom out a little bit so you can see the whole picture So now the printer is trying to figure out what's going on And now you can see the printer is adjusting all the internal components and make sure they're in the correct position Okay, we're gonna do a lot of printers So as soon as I find next one have this at 69 air I'm gonna do it. I'm gonna troubleshoot it on the on the fly So you can see actually how it works, but basically this is what I did I just I just change a change of things on this side a little bit and that areas that are should be fixed That's it. Thank you