 Ladies and gentlemen, good morning and assalamu alaikum. Welcome to the first session of the third day of the STEM education training program funded by British Council. Let's start the session with the name of Allah Almighty. She completed her PhD in biotechnology in 2013 from Government College University Lahore. She worked for three years as teaching assistant and lecturer in Government College University Lahore. Currently she is working as assistant professor and in charge department of biotechnology at Virtual University of Pakistan. Her research interests are in zymology and molecular biology. She has more than 20 research publications in peer-reviewed national and international journals. Today she will deliver her talk on Thermophiles, Biotechnological Potential and Utilization. Welcome ma'am, please proceed for your presentation. Thank you Ms. Samar. Assalamu alaikum. Today the topic of my talk is Thermophiles, Biotechnological Potential and Utilization. It is said that there is no weighted space on the earth with respect to life. Life exists in everywhere where the conditions are not favorable. For example, there are many groups that live in extreme conditions and this group of microorganisms is known as extremophiles. For example, there are many different types of extremophiles, the psychophiles that grow at low temperature, hetophiles that grow at high soil concentration, metallophiles that live at high metal concentration, aceto-philes and alkylophiles. These are the group of microorganisms that live at very low and very high pH respectively. Radiophiles that love to grow at high radiation in the presence of high radiations. Verophiles survive in high pressures and the group that I am focusing today is thermophiles. These are the microorganisms that grow at high temperature. Thermophiles is derived from two Latin words and these are basically the group of microorganisms that love to grow at high temperature. All groups of organisms can be thermophiles but mostly the thermophiles are archaea and some of the thermophiles belong to the group of bacteria and eukaryas. If we talk about the details of the thermophiles, the thermophiles are further divided into different groups that are moderate thermophiles, extreme thermophiles and hyperthermophiles. Hang on the range of moderate thermophiles for 5 to 60, extreme thermophiles 60 to 80 and a hyperthermophiles 80 to 110. Talking about the habitat of the thermophiles, thermophiles exist in very vast different conditions or environments. Some of the habitats are natural and some of the habitats are manmade. In the natural habitats we have for springs, deep ocean basin course, hydro thermovents and petroleum reservoirs. While the manmade include drainage mines, biological waste, fermented compost. Recently, there is a new habitat that is devoted for the growth of thermophiles. This is a phylosphere. This is basically the area of plant that is above the ground. A study reported that when the 16S RNA of the microorganisms of this plant is done, 4 to 99% of the organisms that are isolated are thermophiles that are having a temperature optimum to 35 degree centigrade. Among all the habitats of the thermophiles, hot springs have taken most advantage of the most importance because there are various hot springs and it is very easy to explore or isolate and work on the micro flora of the hot spring as compared to the other localities. In addition to the high temperature of the hot springs, there are some additional factors or additional conditions that make the survival of thermophiles even challenging. The hot springs are often low in organic matter. They contain high dissolved solids, contain sulfur and they are exposed to high light intensity. If we talk about the occurrence of the hot springs, hot springs are spread all around the world from Turkey to China, Canada, Jordan, Malaysia, JAP, United States and Saudi Arabia. Pakistan is also blessed with many hot springs. There are many hot springs that are located at Chitral, Zad Kashmir, Murtazaabad, Hunza, Gilgit, Karachi. And these hot springs are explored more with reference to their physical chemical properties and less with respect to their microbes. Now what are the mechanisms or the adaptations that organisms must have to adopt in order to become able to survive in extreme conditions or extreme high temperature because all of the microorganisms cannot survive at high temperature. It has been seen that the thermophiles, they have high GC content. They have cell membrane that is containing more saturated fatty acids. They have reverse DNA caries. They have increased frequencies of pyrimidine, pyrimidine purine purine composition, histones and histones assembling proteins, post-translational modifications, methylation and specialized proteins, chaffrons. Now focusing on the thermozymes. Thermozymes are the enzymes that are produced by the thermophilic bacteria. Thermozymes have certain properties that they work at, that make them able to work at high temperature. They are thermostable and oppose denaturation and proteolysis. They have improved electrostatic and hydrophobic interactions. They have few thermolabile amino acids that are exposed to the surface. They have exposed, substitution of the exposed thermolabile amino acids and increased numbers of disulfide bonds. If we compare the thermophilic proteins with the mesophilic proteins, as we all know that the three-dimensional structure of the proteins is stabilized by different interactions, that is the ion pairs, hydrostatic interactions, hydrophobic interactions, disulfide bridges and hydrogen bonds. So talking about the thermozymes, they have more ion pairs or more electrostatic interactions in their proteins. They have hydrophobic amino acid residues buried in their core and they have more disulfide linkages and more hydrogen bonds that make them stable at high temperature and resist to denaturation. When we talk about the habitat of thermophiles, it is not usual. There are many additional pressures. There are additional things that make them difficult to survive in the lab conditions. When we want to grow the thermophiles in the lab conditions, we need complex nutritional requirements, appropriate medium-enclosing carbon or additional carbon sources or energy sources. In addition, if we are to talk about the anaerobic microbes, they need additional many cofactors and many micro elements that are without which they cannot grow. We want to utilize the thermophiles in our work or we want to work on any of the metabolites of the thermophiles or their enzymes. The first step is the sample correction that can be done from any of the sources, any of the sources having high temperature. The second step is the isolation that can be done by different techniques, spectrate method, pore plate method or streaking. Then is the characterization. Characterization depends on different factors or it is done by different base. Morphological characterization, biochemical and molecular. Morphological characterization, simply the colony's characteristics are studies in biochemical characterization, catalyst test, oxidized test and then different screening tests are done on the basis of biochemical assays or biochemical tests for which different types of kits can be used such as high remel or QTS-20s test scripts. Then the all these characterization depends on the group of the organism that is present in that hot spring or that locality. But for the accurate identification, we need to go to the molecular analysis or molecular investigations. Different steps in the molecular identification of molecular characterization. There is the isolation of DNA, then PCR specific gene amplifications, then sequencing and phylogenetic analysis that give idea of the, give us idea about the microbe that is present in our sample. In a like other ways, the DNA isolation of thermophiles is also not an easy task as it has a cell membrane that is different from the other mesophiles. So in order to isolate the DNA from the thermophiles, we need additional modifications in the listed protocols. We have to give them more incubation at high temperatures in order to disrupt the cells. And then we have to check this purity. As the yields of DNA from the thermophiles is less as compared to other methods or other microbes. When we want to culture the microorganisms or isolate the microorganisms from the thermophilic environments, only a percentage of the microorganisms, these are isolated. Not all of the microbes cannot be cultured in the lab conditions. So if we talk about if there are 100 microbes, maybe 20 to 30 different microbes, we are able to isolate in our lab. So we can't, we don't have the idea of the complete biodiversity of that region or that habitat. So if we want to know about the complete biodiversity of that habitat, we need to go for metagenomic approach. That is the total DNA isolation and then working on that total DNA isolation, independent of the culturing. And then after metagenomic and DNA isolation, we go for the NGS approaches to check for the thermophiles. Regarding the diversity of thermophiles, thermophiles belong to all the groups. There are archaea, there are bacteria, there are different algae, fungi, all these different groups, they belong to thermophiles. Mostly these are archaea and bacteria that are reported. And in case of Pakistan, mostly the horse springs of Pakistan, they are rich in geobeselium strains. And regarding the anaerobic, they are thermocopa strains reported in Pakistan. As we know that the growth of the micro, thermophilic organism is very difficult. And it's very difficult to maintain the thermophilic microorganism in the lab conditions for the, for the production of our desired metabolites. Though they produce the metabolites when we grow the thermophiles in our lab conditions, they need longer time to grow and longer time to produce our desired metabolites as compared to mesophiles. So if we are going to work on an isolate that is that has an optimum temperature of 70 or 80. So there is our chances of that the microorganism or in which the medium the microorganism is growing, it will dry out during such a long time. Secondly, while working with the thermophiles, we have to repeatedly re-culture the microorganism in order to maintain it in the culture. If we are not going to maintain it in the growing condition, it will become dormant and it's very difficult to re-culture that microorganism. So one approach is to, if we want to utilize the metabolites or the enzymes of the thermophilic microorganisms, one easiest way is to clone the genome of interest into some mesophilic host. If we are going to clone the thermophilic gene in the mesophilic host, we will able to get the desired results at the mesophilic conditions and at high level. So if the steps that are included, including in the expression of a thermophilic gene is a mesophilic hosting, first there are two steps. First is the cloning in our cloning vector, then cloning in an expression vector and then checking of the expression. So if we want to clone that gene, first of all we isolate the DNA from the thermophile, then we amplify our genome interest. While designing the primers for cloning, we use a sequence CAT, ATG at the start of the vector. And a GC clamp in order to protect it. The reason for adding a CAT, ATG sequence is that when it is a restriction site for ND1 enzyme, when the ND1 enzyme is later used to cut the DNA, it will give an ATG that is the starting codon. So it will help in the gene expression. Second is after the amplification of the gene, the gene is ligated into specialized vector. One such vector, specialized cloning vectors, one such vector is PTZ57R by D, the purpose or the benefit of using this vector is that it gives easy screening. It is a linearized vector that is having a T overhang. When we do the PCR of the gene, there is an A overhang at the 3 prime end. So by using this vector, the A overhang and the T overhang then combine and facilitate the ligation of the gene into the vector. Secondly, this vector also helps in the easy screening, that is the blue-white screening. In the blue-white screening, only the clonies that are containing the recombinant plasma or the gene, they are white in color. While the clonies that are not containing the gene, they are blue in color. We can have an idea that the white clonies are having the gene, but in order to further confirm it, whether it is having the gene or not, we confirm it through the cloning PCR and double restriction that gives the idea whether the gene size is obtained on the gel or not. Cloning in the cloning vector helps to save our gene and to increase the copy number of the gene, but the gene cannot be expressed in the cloning vector. As we want to work on the expression of the gene because our ultimate target is to use that gene product into our future research work or in our industries. So we clone the gene into an expression vector. First of all, the cloning vector is double restricted. The gene is then again ligated into another vector that is FAT 22B plus or some other vector. These are also specialized vector that have some adhesion, some deletions to facilitate the expression of the gene. Then the ligation of the purified gene with the help of T4DNA ligase transformation into E. coli. In this stage there is we have to check with the help of activity essay with the help of colony PCR or double restriction because the expression vectors still now do not allow us to rapid screening like the adhesion of dyes as in the case of cloning vectors. Once the gene is cloned successfully in the expression vectors, we will need to further confirm whether our the product of the gene is functional or not. As we are changing the host as we are changing the mechanism of the gene expression, there are chances that there is protein but it is not in the functional form. So in order to check this the recombinant cells are grown in a medium with ampicillin acts as a marker that does not allow the growth of the cells that are not having the plasmid. Then as the plasmids work under IPTG inducer, so these are induced with IPTG cell harvesting is done and then after the cell harvesting there are two fractions. One is the intracellular fraction and then the extracellular fraction. In most of the expression studies the enzyme or the metabolite that is expressed is in the periplasmic space or is in the intracellular fraction. But both of the fractions are checked with the help of STS page or activity essay. Once it is confirmed that the gene has been purified gene has been obtained the partial purification of the of the enzyme is done with the help of ammonium sulfate precipitation. And then final or the complete purification is done with the help of chromatographic techniques. After chromatographic techniques the enzymes are carried out to check the expression of the desired enzyme. Once the expression is obtained and it is optimized with respect to different factors such as the concentration of IPTG time duration effect of metal lines in order to get the maximum production of the desired enzyme. Because of the benefits of the thermophilic nature of the microorganisms and their thermozymes. These are often used in the industry and preferred in the industry for the different functions or different phenomena. Some of the things that some of the factors that make them important or the make facilitate the use in the industry include the robust and far these are the robust and fast growing microorganisms. They have high growth rates these enzymes are the most stable at any because of most of the chemical reactions in the industries are taken out are done at high temperature. So there are chances that we use mesophilic enzymes they become the nature. So using the thermophilic enzymes they facilitate and they accelerate the reaction. And when we use the thermophilic enzyme there is a low risk of contamination because at high temperature normal microorganisms or mesophilic microorganisms don't grow and only those organisms will grow that are thermophilic in nature. They promote the stability and efficient mixing of the subset and facilitate the downstream product recovery. When we have to downstream or we have to purify our product from the cell cell medium. If we are having a thermozyme or a thermophilic product then we simply need to heat that mixture at high temperature. When we are heating that mixture at high temperature all of the mesophilic components or mesophilic metabolites they will be destroyed and only thermozymes will be left. Despite of the advantages these are not very much commonly used because of certain disadvantages as the genetic makeup and the metabolism of thermophilic microorganisms is not completely known. And there are bioengineering tools that are less efficiently used in case of thermophiles as compared to mesophiles. Infrastructure and fermentation expertise these are built mostly around the mesophiles. And these are not suitable for all the heterologous protein infection expressions. Sometimes the expression is not at the level that we want. Regarding the applications, the foremost application of the thermozyme is in the polymerase chain reaction. We all know that the TAC is operated from thermo-sequaticus that is used in PCR and PCR is used in different diagnostics. In addition to TAC polymerase there are other polymerases that are also obtained from the thermophilic microorganisms and routinely used in PCR. Similarly there are certain pharmaceutical applications of these enzymes. Thermocellular proteases are used in pharmaceutical applications, especially the stereoses of thermophilic fungi that produces pterin and pterin has cytotoxic activity against the cells having colorectal carcinoma. When we talk about the extremophiles, it is not only the one factor that is present in that extremophile. Sometimes more than one factor is combined with the other factor and they make a combination of two or three different extremozymes. For example, the metal toluene thermophiles. These metal toluene thermophiles, they grow at high temperature, they are also resistant to high metals. And these are used in biosensing, bio mining and bioremediations. Similarly, the biotechnological applications of thermophilic microalgae and cyanobacterium includes the production of bioethanol, biodiesel, wastewater management, pigment production, di-removal, antimicrobial agents and antioxidant effects. Thermophilic fungi has been used to improve the soil fertility for the production of different enzymes for antimicrobial production, biotransformation and bioremediation studies, composting and biofuel production. As there is a list of enzymes that can be obtained from thermophiles and used in industries, some of the common ones such as cellulose routinely used in paper and pulp industry, food industry, textile, fuel, bear industry, agriculture industry and waste management. Microbial liposies have been used in textile industry, cosmetics, paper and pharmaceutical industry, biofuel, leather industry and detergent industry. Thank you and if there is a question. Thank you. Okay, now we have accepted the thermophile in the beginning. You can take it as such. You can take it as such. One more question. Okay. Okay. Okay. Although the gene is present, we are getting the results on the STS page but it is not expressing because change of the conditions has affected the structure of the protein. So we have to reconsider our vector, reconsider our host, reconsider our conditions, reconsider our inducers in order to get our optimum results or desired results. Yes. In each, in every experiment we have to run the control, first we optimize the conditions at the thermophilic level in the lab scale, then we transfer the gene into mesophilic host, optimize it and then compare its effect. One of the purpose to do the cloning experiment because it is not an easy task. It takes many retakes in order to get a successful result or successful experiment. So one purpose is to improve the expression of the gene or the expression of our desired product in the mesophilic conditions. We need to change the vectors, we need to change the conditions, optimize the conditions, check the effects of different ions, different metals, different co-factors and then when we get the optimum results we can save that condition and that particular vector for future studies. Normally all the experiments we do, all the experiments we do to the students, we choose mesophiles because it is easy to handle mesophiles. The literature that is mostly available is also available on mesophiles. So the conditions of mesophiles are readily available and if we do this then we will get the optimum results with this organism. When we go towards thermophiles, the habitat of thermophiles is completely different, it is very difficult to maintain that habitat in the lab. For mesophiles, a simple medium, whatever medium you give will grow in it. For thermophiles, additional factors are required. Additional co-factors are required. Additional, most of the thermophiles because they are at high temperature, their oxygen level is less. When they come to the lab, they don't grow. Although they are not anaerobic, but they don't grow. For that you have to do optimization studies that you have to check whether it is facultative or obligate. If it is facultative then you have to do different experiments for that or you can use the pore plate method. Or you can use the layer for that so that the oxygen level is less than the routine or normal atmospheric level. So all these conditions are reported in the papers and if it is not more than that then when you work on thermophiles or any extremophiles, then you have to optimize their conditions on your own which takes a lot of time. Yes, my PhD work was on Azad Kashmir's Hotspring the Tattabani. The isolation and factorization of their microbes is on the potential of biotechnology. Most of the robotic microbes are thermophiles. There are different screens for geovacillus and the anaerobic ones are thermococcus screens. No, it is not. The highest temperature of the Hotsprings range from 70 to 75 to 80. The optimum temperature of the enzymes in the geovacillus screens is 70 to 75. The optimum growth in the thermococcus is 80. But for the corticus... No, for the TAC polymerase enzyme you need a full strain thermococcus or pyrococcus which you can use to express the gene. If you don't have a source then you can express it, it has to be in mesophilic. I think CAM generates its own TAC. Yes, they use their springs. It is possible that the strains are also available commercially. You can express the strain commercially and save it in your lab. Yes, it is possible. Because no matter how much pressure I have put on it or on other Hotsprings, most of the strains in Pakistan are geovacillus. This thermo-TAC or the higher temperature of the Hotsprings range from 90 to 85. Initially when I isolated there were about 35 aerobic and 7 anaerobic strains. But when I further characterized on the basis of morphology, physiology and biochemical molecular basis, there are basically 5 different strains of geovacillus and 2 different strains of thermo-poccus in Azat-Mishmi. Thank you. Assalam-u-Alaikum. Can you hear me? Hello. Assalam-u-Alaikum sir. This is Samar here. Samar, Assalam-u-Alaikum finally. I am so sorry. I think this issue has been there due to my... I think some issues were there. So anyway, I am back now. So did you manage to open up my talk? Sir, it's over. Sir, your presentation was tomorrow, I think. Was it today? On the 16th? Yeah, Ashmi-san just started it because I was on the 16th. Yes. Yes, sir. According to schedule your presentation is tomorrow. Ah, okay. Okay, then maybe it's wrong. Sorry about that. I think I was just... I got a message from Ashmi-san that we are not able to open this up. Yes, sir. He told me. Yeah. You will connect tomorrow at 10 a.m. or 10.15. Okay, okay. Well, that's fine then. Please then continue. Sorry, I got interruptions. So please do look at what I have sent you as a recording. So then maybe I'll have a separate chat with you. So my colleague, Abhiya is getting in touch with you. So, because I'll be in the airport. So we are sort of without travel. So let's see if you can see if that is working now. Check it on your computer. Okay, sir. We were downloading the recording you have sent. But I think it's a problem with it. No, no. There's a YouTube link that we shared to you. Yes, yes. So it's not working as well. Did you respond to the, to the, okay. Maybe I think you're talking in front of other people. If you, if you just drop me a text and then my colleague who's helping on this is Rabiya. So Rabiya will be in touch with you. Yes, sir. Rabiya was in touch with me and I told her it's not working. So it is requested to model if you come online at 10, 15 or 10 a.m. We'll do that. So I think it was in my diary is a 16. So this is that pretty sure. So I think maybe something has crossed the wires. So your lecture is of one hour. Yeah, yeah. Let's start right now because our next speaker is a bit late. So let's start today. Okay. Okay. Okay. Okay. Thank you. So sorry about this whole episode. And I really appreciate what I can say. And when you travel, things are not in your control. There's always a bit of trick. It's okay, sir. No problem. So everybody's set. Okay. But anyway, I think I'll just quickly say thank you so much which university for giving me a chance to present. And again, a bit of my introduction, I would say, and thank you to the British console, putting investment into this program. And I represent here up sign as well, which is a UK Pakistan science innovation network. So based here in the UK, but we work in Pakistan. Most of you know another program which I'm engaged is a program about savvy. So savvy is a digital platform that links the research translation to the farmers. And thank you so much again for spending time and I appreciate it. It's not a best practice to deliver things online, but I think we are now used to this due to COVID. So things have moved on. So which is giving us a much more efficient as well. So as I am traveling at the moment, I'm in Saudi. So this is all beauty of the technology and the virtual world so that we are connected. Connection makes us much more, I would say, efficient and much more reliable as well. So I will go in more details about the innovation and enterprise, why it is important in a research setting in a university or a part of curriculum. So I have a few slides here which I will just run through with you and then maybe come up with some questions. Can you hear me? Sir, we can hear you, but the video is not audible. Okay, so the video is not audible. I think there is one option here, share songs as well. So now you will be able to play an important role. And can you hear now? Taking forward the challenges that are here. The audio community is facing it. So I have five components to cover today in our night to define what's innovation and why is the need for innovation there and what sort of innovation processes are and then technology readiness level, we call this TRL. And then I'll just jump on to the business plan. Any questions you have, you can ask me afterwards. I think it's just getting crazy. So maybe I'll use my own sound. So this is the recorded one it's making. So I think the discussion will have more. How many of you know about innovation? So what does innovation mean? Please raise your hands. I just want to see how many of you know about innovation. Okay, quite a few of you know about innovation. So innovation is basically a new idea, a new thought, new idea, innovation. Thank you. Next person, please. Yes, maybe just let me make because I can see everybody. Okay. Yes, please. Yeah. So the next person, please. So when we say new idea, new thoughts. So, ma'am. Actually, new idea with an applicable process or an app. You can create some application or something which is doable actually. Okay. Thank you. That's very interesting because doable. So I think the word doable is important. So you could think something could be imaginative but putting them into practice is the important bit. This is where, okay. There's somebody on the back here raised hand. I think in blue. Yes, please. Green. I think the idea which is doable and sustainable too. This is very helpful because sustainable is the important bit. So sustainable mean where it could be. So maybe just define what is sustainable. So please maybe there's because you have opened up quite interesting. Sustainable is something which can survive, which can stay for a while, or it just does not just go away after some time. It's not temporary. It's there to stay permanent. Okay. That's quite it. You use this word permanent. Permanent is debatable. I would say. Isn't anything permanent in this universe? Things change as well. So maybe sustainability have different definitions. So if a corporate firm, like I'm just giving example from Pakistan context, Nestle wanted to be sustainable. Sustainable in their profits. So what does it mean? So maybe just elaborate a bit more. So sustainable mean not only is. Yeah, what sort of sustainability are. So there are usually three components of sustainability. Anybody know what is that? What are the three components of sustainability? Anybody know it? Sustainability. It's, I mean, always we look at. I just give you example of food. We give example of clothing. So sustainable is three components are always there. First is always the key thing is. What is what benefits it's going to bring to you. So if it's a food, if it's food benefit is in terms of health. So, but if it's. I would say. Important element is economics. So is it economically sustainable? So maybe solution we have today. I just use a simple, maybe the water we have available in Pakistan is a limited resource. But if it's, if you are looking at the same water in a place where it's in the desert, so it's a quite expensive bit to have that water in desert conditions. But also it's a fact on the environment as well. So I think with the important element, these are sustainability is much more linked with the environment. So how it is sustainable. The operations that you are using sustainable for the planet in terms of protecting the environment. So are we using a lot of energy, a lot of water carbon to, to address a challenge or a solution that we are bringing in. So I think this is where sustainability is. Even if you go in future, whenever we look at innovation, it has to be sustainable as well. Sustainable not only in terms of economics, sustainable in terms of environment, sustainable in terms of society. So these are the four important pillars of three important pillars of sustainability. So this is like a triangle. So how to make a balance between society, between economics and between environment, the economics environment and society. So three pillars are three corners of a triangle. So it's basically innovation is always looking at those three pillars are three corners of the triangle. Anybody want to add what does mean by environment here. Anyone volunteer just want to explain from Pakistan perspective what environment mean for sustainability. Ma'am, I just saw somebody was putting their hand and they just put that down as well. Assalamu alaikum, Dr. Fana Lalaro here. According to, I think environment is this drowning in which we live. Yeah. Okay. That's really interesting. And what sort of my years are taken. So when you look at environment and you are looking at innovation. So what should it make to the environment? There are many a biotic and biotic factors. Okay. Which constitutes our environment and interactions among all those factors. Okay ma'am, I just that's very interesting. And I think that's a quite scientific way to explain biotic and biotic. So what does it mean for a common person? Ma'am, I just want to again go back to you. Please bring the mic to the man. So what does it mean for a common layman? So that environment, what does it bring to me? Sorry, maybe my coffee. It is this drowning with which we interact. So, okay. So you are a farmer. So what is the environment mean for you? So maybe just give a bit of example here. So my innovation is going to bring something new to you, but what will be a benefit for you as a farmer? Maybe just elaborate a bit more please. Sorry ma'am, I have the mic off again. As you have told earlier, all these three components means a farmer, his crops, and the factors affecting the crop yield and then the selling of the product. All those is important for the farmer. Again, as I said, environment, economics, and society, everything in it. For a farmer. Okay, okay. I think I just will maybe elaborate a bit more. I think if I'm a farmer, for me, environment means this kind of, it will benefit me. How is my crop health? My crop health is affected by again, things which are coming out of the environment. So these past diseases, humidity, temperature, because that have effect. If my technology or my innovation is going to address the challenge around the farm. So environment would be, environment is more like a public property. We call it as a public good. But sometimes farmers are researchers. We are a bit, we take a blind view to that. These things are fragmented. We've not taken those concentration. So I think important element, because you guys are from the social, from the stem subject. So I think my innovation have to protect my environment as well. So this is the key that we always use in the UK as a yardstick. So is it environment friendly? So if I'm producing, suggesting a technology which is going to use tons of chemical to control one problem. So where is this chemical going to end up? Of course in the environment. So it's going in the air, it's going in the soil. When it's going in the soil, it's affecting the microbiome in the soil. When it's going up in the air, it's affecting the air quality. When it's going, I would say, with the water. So it affects the water as well. So which is an important resource. I have actually another slide, which I just want to show because I was just rumbling about the subject of sustainability. This is more sort of a bigger view. So economy is profit, which is always meant for a farmer. But which is like a micro view. So I would like, if I'm a farmer, I'd just like to see what benefit is to me, the increasing production and making more money out of it. But the benefit for the environment environment is more like a public good, which is of course, it gives benefit to the farmer, but it's a holistic. The farmer has to think about it. As somebody mentioned permanent. So keeping a, are conserving things. So if I say, okay, I want to leave from my next generation, a lot of assets. We always think about properties, always think about cars, but we never, if I'm a farmer, we never think about the resources, which is water. And you will be surprised to hear that Pakistan is one of the water scarce countries in the world. I'm at the moment, I'm in Saudi Arabia. I mean, Saudi Arabia has a worst water crisis. I mean, these places are, some places they don't have rain. Pakistan is blessed with water, but I think the amount of water that we are abstracting from ground. So this is going to be a quite challenging thing. And then the benefit for the society, society is looking at food quality and something benefit in the, I mean, there are places, I mean, even I think I'm not sure in Pakistan, but in the UK, if you buy a meat from a certain region, it's a high price. I'm sure in Pakistan, there is a, like Basmati, if you say, the Basmati of Gujranwala, the price will be something else. Instead of the Basmati born in Multan, the Basmati of Multan will also be Basmati, but the Gujranwala is a benefit of the society. So again, something very specific to the society. So I just mentioned climate change, water, salinity, health, food benefit, past disease threats. This is more like a bigger challenge that society is facing. So, but price fluctuation, I mean, you are in Pakistan, we see in good pridia, so if the price increases in Pakistan, then people come to the streets. It's a global thing. Because again, if the farmer, if your innovation is going to help the farmer to produce crop more expensive, this means it's going to be more expensive for the society. But if your innovation is going to help the farmer to produce more crop but cheaper, high in its quality. So I think I think quality should not be compromised. I think this is where the biggest question is and how much chemicals are used there. So that's something have to be kept in mind as well. So I think when are we looking any innovation around the farming sector or in the food sector or I would say in the life sciences. So these three things are paramount. So we have to look at them for the efficacy. So I'm just going to stop this one and come back to the original slide, I was talking earlier. So I'm and I think the other element which is important in this debate of innovation is doable. There's something you can do. I think one of your colleague mentioned earlier here in the insure way I work, we have developed the technology but that requires something is about. So what is innovation? So innovation happened, nitrogen inhibitor technology but that would require two tons of that chemical that chemical is basically a plant based product that you can use, but that's something is in scale-wise it's not doable. I'm going to the next slide. Thank you. When someone improve the next slide. So we're saying from eminent people, especially Thomas Edison, who you all know is one of the innovators who done interesting innovation. He said there is a way to do it better, find it. So there's always, it's a continuous journey to make improvement the way we do. So I think the world and we all know the famous. The last one was, there's always room to improve it. Do things in a different perspective. So I give you example of, you know, a Fuji was quite popular many years ago. Fuji was popular. But I think these guys did not do much innovation. We have done one product is sufficient to go forever. It's not the case. So Fuji is nowhere now in this camera technology. So maybe they are all gone into different perspective. Motorola, these companies, Ericsson, these guys were just wiped out because the new product has taken their place. And that product has again a shelf life. We call this each product, each innovation has a shelf life. So shelf life is how long it can continue. The one thing in the scientific world, we use this patents. So you can patent your product, but I think this is again, patent cannot be granted forever. So they're only granted for 20 years, but it costs you money to maintain them. So once the patent is filed, you need to exploit it and get to the market. Otherwise after a few years, I mean, I think there's a lot of pressure now. So companies are saying, let's bring it to 10 years or maybe it's short duration. When you file a patent and it takes you three, four years before it's being granted. So first, if it's 20 years, five years are already gone. So you have only got 15 years. So now there's a lot of pressure that we should reduce it to the next level so that the new companies could jump in. So this means you cannot protect yourself. So this is a continuously. We all know that Albert Einstein, he said we cannot solve our problems with the same thinking we used. So the previous thinking or the mindset cannot continue. So we have to create something new. So we have to keep on looking at the new explorations. And we all know the famous saying from, I think I'm just going to the next one here. And the last bit here is, we all know Steve Jobs, who was the innovator. I would use the terminology was innovator. I use the, who helped us to develop the Apple phone, Apple watch, and all these Apple, I would say. So he said innovation is the ability to see changes as an opportunity, not a threat. So it could be a new opportunity that could help to solve the problem in a better way. I think I'm just going to maybe stop for a moment. Maybe ask again the group. Sorry. Sorry. Just maybe. So some in. I'm sorry. I'm just going to. So innovation. I think I'm not going to use this recorded version because it's just annoying me. They just want to ask here at the group. Maybe you guys could appreciate me. So maybe give me one example of an innovation in Pakistan, which was just on the top, but then it's just gone to ground. Anybody can give an example, please. Yes, please. Anyone have any example, please. Gigi, please. Any story for Pakistan or any. Any cameras. Different types of Microsoft's. These are the ideas. Sorry, please. I'm just saying about anything which was stopped and gone down. Okay. Shoes. I don't know about that. In mentions. Problems. For the blind people. Okay. Okay. That's very interesting. But I think. What do I just want an innovation? Again, innovation. I want to add something about BT cotton. You must have heard about it because you know that Pakistan is an agricultural country. So our economy mostly depends upon the products which are we get from the agriculture. So BT cotton. I think this is an innovation in Pakistan. Still, the farmers, they are growing the BT cotton in different areas. So I think this is an innovation in Pakistan. Farmers, they are growing the BT cotton in different areas where cotton is growing. No. BT is interesting. But I think that's again a, we call it you go to the, you set up a quite high milestone. We missed something in between as well. So now there isn't any further improvement going from here to above. So the BT was the, I would say, this is where the things are now progressing as well. I think that's an interesting example, but it's every innovation has a peak. So it goes, the dot con has a peak and that is came down as well. So I think the point which I'm trying to say and again in the research is there's always a gap. So we cannot simply say, okay, this one, okay, Apple is end of the world. No, Apple is not end of the world. So there are things which are still coming on. And again, all companies they're not making. So with this terminology, we use open innovation. Have you known anybody open innovation? What does it mean? Open innovation? Anybody want to say open innovation? Yeah. Okay. Ma'am, anybody else? And this concept is now used by the companies, a large companies, multimillion companies, Google, Facebook. So they do not say, okay, I mean how many people they can employ? There's always a limit. So you can have maybe thousand people, five thousand people. But in a daily job, your mind is doing things which are, you ask to do. So you are confined. But if you're looking something outside the box, so it's always, this is where these innovation companies are saying, giving their employees, there's a lot of open and flexibility. Work space, you go and sit in a cafe type environment, relaxing environment, where your mind would start thinking about those ideas. But again, there's a limit. So they say, us investing on innovation, let's let the public to come up with ideas. So the companies say, okay, we would be open to take ideas from outside. So they organize events, they organize workshops, they organize those opportunities and engage with the public. Public mean researcher with the universities, which are not on their payroll and their innovation, they can pick it up. So now, if you look at Apple, Apple may be doing its own things around 40 to 50%, 60%, they're outsourcing stuff from others. So this outsourcing thing itself is called open innovation. So the, which you have opened the doors for others to contribute and bring it together. So it's like a funnel thing. So you get, let ideas shift them around and then bring it to a something, which is useful. So we, where I work in this Institute, we, I think three years ago, we arranged a big event, open innovations we get. And again, this is a bit of a marketing word. Today is not any word which is open. So open is always coming with somebody who owns it. So this is where I think the concept which universities and academia could promote is problem solving. So when the problem solving could be done by students, I mean, I mean, in the Western world, we use a lot of time feedback. So when you get a feedback, feedback is basically taking those ideas and then you find in your product. So the open innovation is basically this taking the, this feedback approach, getting others to contribute into your products. So which is again, a good way to look at innovation that could make a difference as well. So I'm here actually at the moment, spending some time in the Saudi Arab, one of the big universities called cost. So cost is a university which is established by the, the kingdom back in 20, 2009 or eight or nine, 10, but it was operational only 2013, but in less than 10 years, this university has become the word second largest, our second top tier university in terms of ranking. And they, what did it, this open innovation, they just let the word to come. So they, okay, you have any idea if you come to us. So that's, I think that's a fantastic example, cost is an example of open innovation. So they get, they got people from Japan, from the US, from the UK, Germany. So they pick them up, bring them together. And then when these minds sit together, the ideas generated. Another example I just want to give you is the bottle of water. So I think we, we use this terminology. I don't know how many of you know, discipline, interdisciplinary. So can somebody say, what does interdisciplinary mean? Anybody would like to volunteer interdisciplinary research or interdisciplinary, remember the black, please, sorry, my color recognition is just gone. When more than one departments are cooperated and in the cooperation, they have to work. Then it is interdisciplinary. Excellent. Excellent. Excellent. Excellent. Any example you want to mention? I think interdisciplinary research means the integration of two different disciplines into a single research purpose is known as integrated one. The example is, let's say the scaffolds that are the replacement for the knee cartilage. These scaffolds are made by some engineers. And then they are biologically tested and biologically filled with the cells by some biologists. So this type of research is known as interdisciplinary research, I think. Excellent. Excellent. Anybody else? An example of interdisciplinary is the stethoscope. Some physicists actually invented them and biological. In medicine, we are using the stethoscope. Okay. That's very interesting. Yeah. Anybody else? So I think you have explained. So we just give you an example. This water. Water is a discipline itself because water is inside. I'm sorry. Maybe just a simple water without using this bottle, I would say. So and then the next thing is I would say maybe water is not a good example. I just kind of okay, I'm going to just apple. Apple is a discipline. One discipline. So you chop this apple and you cut this into a fruit. So you chop this apple and you chop this into a fruit. So you chop this apple and you chop this into a fruit. And maybe mix some other fruit like maybe I would say orange. So this has become a bit of a week. What do we call it in Pakistan? We call it fruit chart. Fruit chart is much tasty. It's more appealing as well rather than just apple. So it's more appealing. So when you bring two disciplines, so things get quite interesting. So when you take to the next level, so you have apple or in orange banana mixed here. You make a milk shake. Take a just for your smoothie. The smoothies is something you can carry along. You can put into the bottle and you can drink it when you need it. So it's going to be okay. Apple key value to increase. Okay. Come to the next level. So I can know this the apple and orange and the banana. These three things have come together to provide a solution to one problem which is thirst or energy or if you are going to do a marathon, something you want to keep with you. So that's something that's interdisciplinary. So you bring various disciplines together to come up with a solution which is a smoothie or a juice. So I think the size with the way it's going at the moment is inter-disciplinary or trans-disciplinary. So where you bring sizes together. So otherwise the guy who's working in the computer science department, they were just doing it maybe morning. But now this computer science people are working with a biologist and they are working with a mathematician. So they are coming up with a solution that is trans-disciplinary which provides solution to the next level. And I think this is where the word is going. And if you now do any funding proposals in the UK, you need to have people from different disciplines. Not only science, you need to have people from the social science. So you have it done a fantastic work. Your products is fantastic but it's not being communicated. It's not coming to the to the means to the public. You need to have a sociologist. You need to have a columnist. It's cost-effective. But I think this is where things are going. So okay, I'll come back to again my talk. So I'm just getting distracted guys. So please if you have any questions just raise your hand any comments. Anybody want to say anything here on this subject ma'am? Hello Assalam-o-Aliakum. Sir, I want to add on that every I think applied sciences or applied research is interdisciplinary. I mean, if you want to apply an idea and you have a gain some product then obviously without the involvement or even for the formation of the project without involvement of other people, you can't complete it. For example, if there is a waste management system or waste treatment system, you need engineers, you need environmentalist, you need the study of the hydrology and the water sciences. So multiple people can work together to make something effective. So this is again interdisciplinary. This is applicable to any applied area. So if the innovation, the thought is converting into the wealth in terms of the applied product, then we can say that everything we are producing is interdisciplinary without the involvement of other people. We can't complete it from the other disciplines. I have worked on the hydrogen base membrane biofilm reactors for the waste management and then we were having the mathematicians and then engineers and then obviously environmentalist and microbiologist. So these four sciences they work together to make something effective. And now I have also produced one PhD student and she has been working on the bioflogs. So among in this we have connected the microbiology and the aquaculture and we have made a sustainable technology. Bioflog is well established in the Malaysia, Indonesia and other countries but in Pakistan it is getting fame during this decade. So we have also reduced the water loss. We have managed the toxicity in the aquaculture so with the least consumption of the water and by using the water waste as a food for the fish as well as the microbes we are making the fish. We are growing the fish. So these were my comments with respect to the innovation and interdiscipline sciences. Thank you. That's wonderful. I think I love it. I think this is there. This could be a case study. Bringing people of different disciplines and then again you bring these people, you bring expertise, you bring the human side into it. So we use this terminology in the UK and I think something is quite famous in the development in the development sector. Human centric approach. So when a HGC, when you are human development centric approach, whenever you are developing a solution or technology it's good that you test with the people who are the end users. So see what would be their view. So what if it's going to bring to them which pains it's going to resolve, the challenges it's going to resolve, what would be their convenience as well. So what availability has a problem to resolve. This is fantastic. So I think this is the way the innovation have to bring that focus in mind. And I think 10 years ago people were just looking at silos. People are looking within the department, people are not talking to each other, even within the department, they are just talking. Mathematicians, physicians, people in the I think you need to have being, did you bring them as sociologists as well into this project? I think I don't remember I was working with a scientist and they weren't included any sociologists at that time. And even in my PhDs during I think we haven't involved any sociology but ultimately it is going to So I think sociologists will also be needed. I think this is important. I think sociologists are a critical role. So I think that something must be better done. The society accepted. Exactly. Thank you. Okay. I'll just quickly go back to my. So we call it this innovation could be in anything. This is just a few examples in a process. So for example, enabling process. Core processes, technology-wise, in terms of its offerings, its performance, product system services. And if it's a delivery, so all you deliver your product. So something is delivered through distributor, something delivered through online, something delivered through, I would say, peer-to-peer, like these lectures. This could be innovation as well. So instead of having a physical, you have virtual sessions. This is a new way of delivering things. In terms of finances as well. So is this innovation going to be creating a new business model? Who is going to pay for it? We always use this terminology, big data. Big data is an interesting example. So Google knows now what we need to do. So Google knows. I mean, sometimes I park my car and says, this will take how many minutes to get to home. So because it takes your location and then you next information. It's a new model. That information is providing some kind of maybe a, when my location is shared, maybe it's going to the Tesco or my local store. And then when I'm opening up my computer or my app, it's showing me some, I would say offers from Tesco store, because I'm quite close to Tesco. This is a new business model. So new way the things have started moving in. And then we call this networking as well. So I just give you some examples. Okay. So next. So innovation journey. How does it work? So innovation basically is a mix of creativity and your ads on commercialization. So when these two things come together. Okay. So we just, I think we touched on this already. So we just need to skip to the next one. So. So some examples of innovation in the past, you say how interventions have helped to deliver innovation. We all know the civilian thing. This was many centuries ago used for grinding grain to make bread. So now that's been used by everything in this world. I would say in terms of that runs on wheels, by cars, by airplanes, by turbines. So that wheels, a wheel concept is used in a variety of functions. Here is the processor microchip, which was maybe originally developed for a computer, but it's now used by processors all the way from sensors. And in opening up keys, a car, you're using your sensor for that. So which has got, I would say, this motherboard. And use of laser light. It's not only sort of used for fancy reasons, but it could be used to number of things from printing all the way to doing cutting edge. I would say the work in the manufacturing. So I think we have. So innovation took many years sometime. So we all know this example of mobile phone. So I think I just need to skip this one. So how and when and where the innovation starts. So it's always a start from the conception. So from an idea. So what is the need someone is trying to help. And once that idea has been tested, idea is generated, it's been worked out, it's planned through. Then you are thinking about how you would like to implement it. So in terms of product development, product testing, we call this prototype testing, prototype development, prototype application, pilot application, and then testing. So one is tested, then you like to take to the next level and bring it to the market. So when you are looking to bring in the market, it will again require production. And the market would be tested again and see. It's maybe it's local product starting in one particular area and then take it to the next area. And then countries nationwide and international as well. So three steps, the conception. Anything you want to add here, guys? Any comments and any suggestion on this slide? No? Okay, then we continue because I'm just conscious of the fact I think we need there's new lecture starting soon. So I need to wrap this up for you guys. Okay, so the next one is the process. Sorry about this is quite complex chart here, but just the whole idea is just to say it always start big. So the new product idea always have many things that you like to bring in. But then it's been cut short in the product concept stage. And then it's been tested in a next level. And when it is tested and it's just been going through a bit of a funnel approach. So coming down to a and this idea is when ideas new, it's always the early doctors who are doctors, not everyone. So I think so this is a bit more detailed. I would say innovation journey itself. So where does it start? We always say it's like funnel report is always start big. So you're looking at sort of the bigger markets where the idea would go where the innovation would go. And then you trim it down to the next level and then you go through the process. Okay, I cannot do this. I cannot do this. Maybe bringing it to the next or it's a precise level. And then maybe you original idea was maybe 10, 15 different attributes. But then when you bring it to the final stages of focusing on a very particular advantage that it will deliver. So I think the important bit here, which I was going to say, I think because of this video is creating me a bit of a, I think one thing I just want to explain here, the one technology, the idea, but now stage difference is over. So super different stage, but like you have to week in scientific word, you know, what do you know about that? So just TRL. Anybody? Yes, sir. Any suggestion about TRL? What is TRL mean? Technology readiness level. Raise hand, anybody, please. What does mean technology readiness level? Ma'am, tell us about the hydrogen. Okay, sir, you're talking about basic technology research. It's a technology readiness level. This is obviously the one thing which is so first study at the bench level in the lab scale and then obviously the pilot scale and then it goes to the industrial scale. So among these skills, they are different levels. So we research, we do research, then we make something and then we apply and then we take the feedback and see. And then obviously it is ready to go to launch. I think this may be telling about the technology readiness level that is now perfect to go into the market or to get commercialized. You're right. You're right. I think we call it levels up to nine. So from one to nine. Okay. So as you mentioned that hydrogen base, so it was the US lab and then they were working treatment plants of water and then we got the sample and we got the problem from there and we solved in the labs and then it was the solution were given to the directly to the treatment plant. So I think that was level TRL nine. Okay. So if it's something which is coming out of the lab, which has been tested, it's been... It was applied and then there were certain problems. So the problems come to the labs and then we do the research and get the solution to the problem and then those solution was given to the industrialists. So I would say if you are going some work on a theory level, so if it's something that's still research, master student is doing research and then a lot of work is done to test maybe certain attributes of a chemical without knowing its benefit to the bigger problem. So I would keep that. This would be seen as under TRL four. So anything which is the research, primary research, which if it's a close kind of set, we call it TRL one. So you don't have to bother about market. You're just looking at the scientific side of it. But when you are bringing to the TRL four when the idea is tested, this is something is a proven principle, then it's going into this stair case. I would say next level technology development prototype technology demonstration, looking at on the next level subsystems development and then something which is now a product which has been tested on a small group. It's now available for a bigger people. So that would be a place which is still not commercialized. But I think this is how I would say TRL sits between TRL eight and nine. Okay. So yeah, so once you have done a product, how you would like to bring it to the market. So we all might have heard this, this acronym or this is a quite common terminology used in the business world. We call it business plan. So you have interesting idea. Do you have a business plan? So business plan, what does it mean? So how? Okay. I'm going to stop here. People, you know any business plan, business plan terminology. How many of you know about business plan? Please put raise your hand. In front row, I'd like to ask you. Sir, what is a business plan? I am Dr. Tahir from Punjab. Actually, you are talking with a business plan. So in a scientific terms, as you explained that if we develop something new technology or new inventions. So we test in different levels and then we transfer into the society or a market. But we need to the some persons which is from the marketing like economics or business. So they help us because as a scientist, we are not familiar how we do the business. Sometime we make something in the lab. But unfortunately, due to some hurdles, like testing agencies, some governments, lessons and also the big markets, which have already working business in the society. So it's very difficult to sell our products in the market. So I think it's different aspects because the economics are a little bit different. But in a as a scientist, it's a different things. So I have. Thank you. I think this is the thing. I think the whole idea which we are saying is exactly. So the scientist is good in science. But when this science has to develop a product or a service, then you think about a business plan. So business plan, what sort of components it has. It's always just just a second. I think maybe I'll just. Yeah, so once you have done a product, how you would like to bring it to the market. So we all might have heard this. This acronym or this is a quite common terminology used in the business world. We call it business plan. So you have an interesting idea. You have a business plan. So business plan. What does it mean? So how how your idea is now being materialized and it's going to deliver benefits for the society and what would be its cost and who's willing to pay for it and what would be the revenue model and who would do it, who would deliver it. So it's a it's like a some kind of x-ray of your idea. So it has typically seven components. So one component is executive summary. So which gives the headline of about the business plan and the idea that you are trying to deliver. And then it has market research element into it that covers a bit of research. Second is defining product product product and service. And then now the third element is strategic analysis, typically called SWAT analysis. So that's carried out. And then marketing plan is worked out. So who's going to to benefit from the product and how you're going to sell it. And then operation plan, where the product will be made, where it will be delivered, some distribution plan and is it something, a soft product, is it something digital platform, something available on the internet or on the app. So those kind of things. So who's going to do it team typically. I'll go in more details in the next slides and then financial plan as well. So I'll go one by one out of them so that you can understand this. So first of all is the executive summary. So executive summary is the explaining the key innovation of your product. So our service that you're trying to bring it to the market. So it should have a bit of mention of what is the USB we call unique selling point. So what is additional feature this product is offering compared to other products. And who are the key customers of this product as well. And maybe a bit of a market, a hobby is a market. So and also these days is important that whenever we are trying to deliver a business, it should have a component of if it's addressing the societal challenges. So it's poverty, something. I mean, I'll just go in more details. Sustainable goals. I just have 17 of them here. So we have to see our product is able to benefit the society in the long run. And this will improve our reduced poverty in the area, improve if it's any bioproduct or something which is as a health initiative, something that can help to address the issues around health. If it's a new tool that help to address education gap, so that can help improve literacy or improving the quality of education, improving the gender issues. And important element here is water. So if it's something around climate change addressing the use of water and clean air, clean energy as well. And so these are the 17 goals. I won't go in one by one all of them, but I think this is important that when whenever we are thinking about that this product is going to benefit. So what benefit it is going to deliver to the society, to the societal challenges, to the global challenges we call these days. We call this terminology global challenges. It's been used in the UK a lot. So actually in the UK, they have a program, Global Challenge Research Fund. So that was basically focusing on the UN sustainable goals. So we all know the climate change is happening and the issues around net zero target that we have to achieve. So greenhouse gas emissions, water. So Pakistan has a perfect situation of all these problems at the moment. Again, this is a global problem. Energy prices and air quality and all these things. So we had a recent lead issue with COVID. So COVID itself was a fit in the UN sustainable goals. So it was one of the issues that has caused the global challenge. So how we do it? So first of all, in the business plan, the important element is you do the market research. So what is the size of that market? So if I'm going to bring a vaccine for COVID, I would have to think, okay, what's the big market? So seven billion people, eight billion people, nine billion people. And how many will get affected? So if my vaccine is going to be different than others, so what USP has, it doesn't give you a fever, it doesn't give you any side effects. All these things, if those benefits are proven scientifically, then you can claim it. So but I think there's something you have to do a bit of market research. So how big is the market? Our government regulations are there. Is the freedom to operate? Can you deliver something? Is it something safe to the society, safe to the children, safe to the special segment of the society as well? We call sometimes, if you are in a Muslim country, you are delivering something, you have to have a halal as well. So that's just one example. So the next one is you define your product and service. So what attributes that product carries in terms of its benefit to the end user, its convenience, it's better than others. So we always look at what is the unique selling point, USP. And of course, when you are looking at this product, so if something you are making it, is it something will not be copied by others? So you look at its IP as well, intellectual property. So you protect your patent. So usually, if you are publishing something, you're not publishing if something is new. So you have to do the patent first and then get it published. Otherwise, once it goes to the public domain, it's not be there. And usually the patent is protected for 20 years, I think. So it should be open afterwards as well. And there is a cost associated with patent in the European market and the American market, global market, Chinese market, Pakistani market. So I think this is important that you think about that product, protect it. And then you look at SWAT analysis. So you might have heard this terminology is pretty common. So SWAT is 10th, weakness and opportunities and threats. So strengths are always, so what strengths that product is bringing along. So the business which will give advantage over other companies or other competitors. Weakness is the characteristics of a business which make it a disadvantage. So something could be always a worry for you. There's new innovation going to come or something. It's not, this is always a challenge because one product cannot always offer everything. You just have to just take which are the positive sides of your product opportunities. So it's a element in the company's external environment that allows it is formulation and implementation. So basically you're looking at maybe just give you example of if in Pakistan these days a lot of people talk about CPAC. So the CPAC would be an opportunity. Okay. Sorry to interrupt you. So the fourth element is called marketing plan. So you need to look at. I think. Could you please wind up it? We are getting short. I think I'm done. So I'm just going to say quickly. I think I'll just, most of the things. I think the important bit which is at the end is the product has to have to price, marketing and all these things. I'm just going to stop this one. I have already shared this on the YouTube as well. You can watch it from there. But any question you want to ask? But it was such a good play talking to you. And thank you so much. Sorry. Time was a bit of an issue for me. But we managed. So thank you so much. And with that, thank you. And good luck. Thank you so much. Thank you. Ladies and gentlemen, we will take a quick break of 10 minutes and we'll get back at 12. Insha Allah. Welcome back. Ladies and gentlemen, our next presenter is Dr. Tahir Mahmood. He's associate professor of molecular biology and biochemistry at center of applied molecular biology University of the Punjab. He got overseas merit scholarship from higher education commission of Pakistan and completed his PhD in biochemistry and molecular cell biology from Institute of genetics and molecular and cellular biology University of Strasbourg, France. He also enlisted as a productive scientist of Pakistan in 2017. Today, he will deliver his talk on our new two articles are promising future for health and new drugs recent development and challenges. Welcome, sir. Please proceed. He's family come. Thank you for nice introduction. So. Today I'm going to discuss the one of the important topic. Is the nutraceutical are promising future for health and new drugs. What is the recent developments and the current challenges to convert these new technicals. As a neutral pharmaceuticals. So I'm hopeful you are familiar about this right. Nutraceuticals are those participants who are from the botany from zoology. I'm guessing maybe some of from biochemistry or chemistry. You are familiar this word. Nutraceuticals. So if we look the simple definition of these are basically foods. Or a product that basically provides medical or health benefits including the prevention and treatment of diseases. These two things are very important to prevent and treatment of diseases. These two things are very important. We can use these nutraceuticals to prevent from diseases. And also if the diseases happen in the human being particularly then we can treat by using these nutraceuticals. Nutraceuticals are basically contains the function foods. Nutraceuticals is that means they are from nutraceutical functions they could be functional foods. And what is the functional foods? Actually the functional food is a food that exerts health properties beyond the traditional nutrient that it contains. So these are the functional foods. So functional food ingredients are responsible to show these nutraceutical properties. Nutraceuticals has many advantages over the medicines because they have less side effect and they are very compatible to the human body. Because they are lesser or they have no side effects so they are good for the use of the human beings. With the passage of the time if you look the nutrition science has been changed. Now instead of traditional nutrition science it is converted into optimal mutations. So what is the traditional mutations? It is consumed food and nutrients that required for the growth for the development and maintenance of the body. These are called the traditional mutations. What happens now? It is a move from the traditional to the optimal mutations. In optimal mutations it also provides to promote overall health improve physical and mental performance and the third one is important function of the optimal nutrition is reduced risk of diseases particularly the cancer obesity and cardiovascular diseases. So we can prevent by using the optimal mutations to prevent from these dangerous or very life threatening diseases including the cancer obesity and cardiovascular diseases. So if you look the functions of the functional food the functional food has basically three major functions in our body. The first one is to provide the nutrition means supplements for the human beings. The second one is the most important is the organo-laptics or socio-calcium properties as we are familiar with some dishes, some food so these are the organo-laptics that is commonly used in our society like Pakistan and these two properties are known as a traditional food and if we look the third one added value which is the basically health benefits these three are collectively known as a functional food. So functional foods are important to provide the prevention and treatment from the different diseases. If you look so many years back the founder which is a great physician he knows the father of the medicine several centuries ago he said let your food be your medicine means that you should use your food as medicine So this philosophy actually contains the prevention is better than cure because if you use some specific food so they can prevent from diseases and that can prolong the human life and we are more comfortable more physically fit and mentally fit if we use the good one or balanced food. So functional foods started particularly in Japan Japanese are the responsible to develop these functional foods in 1984 a Japanese ad hoc research group under the scholarship of Ministry of Education Science and Technology started a large scale national project to explore the interface between food and medical sciences So a category of food means food for specified health uses was established in 1991 So the first time functional food used in 1993 by the Japanese and they published paper in nature news and with this headlines that Japan explored the boundary between food and medicines So they have found out what is the components that act as a food and what are the components that act as a medicine So they explored the first time these boundaries that is present between food and medicine Here in this diagram you can see the functional foods If you look at the functional foods So major source of functional foods is the plants and fruits These are the major vital source to provide the functional foods and nutritional products Here you can see many examples from the different plants like rava, orange, grapes and oligurus and different plants of fruits are responsible to provide the functional foods and nutritional products So here if we look the bigger class of the compounds that are isolated from the different sources and they are the plant sources vegetable and including the fruit sources Here you can see the class of compounds for example alpha carotene It is obtained from a carrot and its function is to neutralize the three radicals which may cause damage to the cell So this beta alpha carotene comes from the carrots Like other compounds beta carotene these are also the source from the color foods pigments and yellow color fruits vegetables and this also provides to neutralize the three radicals and which can cause the prevent to damage the cell The other compound is a lutein This is obtained from the green vegetable reduced risk of reduced risk of prostate cancels reduced risk of reduced risk of muscular degenerations and another compound is a lutein which are obtained from the products and it also reduced risk of prostate cancels So another compound class of compounds which are phyto sterols and also sterols they are obtained from different sources you can see from a corn, wheat, vegetables and they are also responsible to lower the blood control levels So another class of compounds which can obtain the omega-6 and omega-3 fatty acids DHA and posa pentanoic fatty acid these are obtained from different seeds like flex seeds it's a hemp, canola oil and some other fish things omega-3 and omega-6 fatty acids and these are the responsible to reduce the cardiovascular diseases particularly because they can reduce the cholesterol levels and also the bad fatty acids which is present in the blood So another compound of the class of compounds which is a conjugated lutein acid that's obtained from the wheat cheese products and these are also responsible to reduce the cancer So these are compound of classes which can obtain from the plant sources from fruits from the vegetables So these compounds are very useful to prevent from the diseases and if diseases happen so we can take in a large amount they can use as a treatment So here you can see another class of compounds is the coals these are these are the big class of the compounds which can be isolated from seed oils such as the canola and sunflower and some other cotton So these are the responsible to prevent the oxidation process that happen in our bodies the anti-oxygen and increase the oxidation to work for the heart and good for the skin So another compound of the class is the cannabinoids and polyphenols they are also present in a large amount of plants and vegetables and in different sources particularly the plant fruits vegetables leaves so they contain the polyphenols and these polyphenols are some of the polyphenols are mentioned are catechic acid phyleric acid, chimeric acid vanilla acid these all are the polyphenols that are obtained from different sources like tea wine olives, chocolates berry fruits etc and these polyphenols is particularly act as anti-oxygen and they are good to prevent from the cancer and they are also good for the heart diseases good for immune systems and they are act as anti-microbial activity these are reported in our literature these polyphenols are working efficiently So another compound is a lignanes and pymons these are also obtained from different foods like fluxy cereals, soybean cabbage and these contain the class of these compounds and it can also act as anti-oxygen and their functions are possible to prevent from the cancer and act as a radical sequences which are generated in other cells they can bind free radicals and prevent cell damaging So another class of compounds is biopeptides these are basically small chain of amino acids they are also present in different vegetables like bean, wheat, rice palm kernel oil palm kernel etc and they are good for the immunomodulator activities So another class of compounds which are isolated from different plants from vegetables, from fruits they are the prebiotics and probiotics these are also accepted from different grains, onions and purified foods and they are also responsible to improve quality of intestinal microclure and gastrointestinal health So these all are the compounds, different class of compounds which are isolated from different sources, they act as a new personality class So here the one slide I want to explain to you how you can extract these bioactive compounds which are present in different vegetables like plants, like fruits bodies different like citrus family fruits So you can extract the bioactive compounds by using the number of techniques are available some are conventional techniques and nowadays some are modern methods are available to extract these bioactive compounds So first of all you extract the source from where you want to extract the compounds then that source can be identified by the botanist either you are having the same species or anything you want to extract either it is a right one or it is not because there are lot of species are available so you can identify and then you write the materials and then you can use number of solvents for the extractions these are the conventional methods still is a good method although nowadays it is little bit creating problems for the environment because these solvents when evaporates goes into the environment they have a problem now we have some other green which are commonly used to extract these bioactive compounds so you can choose the solvents on the basis of nature of your compounds but compounds you want to extract so you can choose the solvent either you want to the polar solvent or you want to non-polar solvent or maybe the green solvents like carbon dioxide water glycerin or some other methods now it is called hydroponic media is available that you can use and extract the compounds so choose the solvents it is a depending on the nature of your compounds either they are polar either they are non-polar like a methanol, ethanol, chloroform or maybe you can use the n-hexane but either number of solvents you can use depending on the nature of your compounds and your interest what you want to extract basically so normally you can try the material and after the bio-material you can add the 1 ratio 10 you take a 10 gram of your solid branded materials plant and you can add 100 ml the solvent and keep it 6 to 8 on the orbital shaker and after that you can basically build samples you can extract 3 times and build sample and then you can basically evaporate the solvents under the using the rotary evaporator under reduced pressures and you can recover the solvents and extract the crude extract and that crude extract you can be identified by the HPLC and some other instruments you can check either your interest compound is accepted or not and then you can check the activity that compounds are biologically active they have some functions so you can use the number of techniques which are available to purify your compounds so purification is also very gross time taking very laborious work but nowadays some advanced methods are available like HPLC LCMS you can use that advanced method and purify your compounds and identify structure by using number of techniques which are available like HPLC GCMS and NMR you can identify the compounds so another class of compounds which are isolated from the different vegetables from our foods proteins from the flowers is called essential oils essential oils are the natural aromatic compounds in the feed, bark stem, weed flowers and some other parts of the plants these essential oils is present in the large number of flowers as well and some leaves also contain the essential oil as you can see there are some smell and that contains the essential oil so this is another big class of the compounds which are present in the plants and they act as medicines so it gives their distinctive smell and provide plants with the protection against predators and business and play a role in plant pollination. Essential oil the plants may involve them to protect from the insects from the diseases and that essential oil you can extract and use for the human beneficial plant. So essential oils are non water based phytochemical made up of volatile aromatic compounds so these compounds are the class of compounds which is like perpins, perpinoids hydrocarbons. These are the essential oils which can be extracted from different sources and it can be used as a nutraceuticals. So they contain two essence of the plant it was derived from essential oils are highly concentrated it does not contain any artificial substances like a perfume and fragrance oil. If you can perfume or a fragrance oil in sub-majority approximately 1300 chemical use or unless it's just 4-5% natural sources and other synthetics. Synthetic chemist they use but if the essential oil they contains the all natural they don't have any synthetics so we isolate from the different sources very good oil the orange all the orange species have a very good yield of oil which is used for various purposes especially for cosmetics which are available so they are very good for the skin health so orange are produced very well they have a very good source like you can explain in my class and they contain a large number of important components which act as a medicines the organic chemistry the basic information we obtain from the plant sources and then they can synthesize in the laboratory. Citrolyte A orange oil is very common available it is used in cosmetics and then diluted with water and ladies use a lot of lavender oil and the skin orange oil is also very good for health you get from the Pansar shop and often ladies mix it with honey this is also a benefit because we have waste materials because we have a lot of orange peel you know Pakistan is not a very big producer in the 10th and 11th we produce orange but all the peels are very expensive because their yield is less natural source they are very good for health but there are such components in the health in the skin and the black spots they recover so natural oil is healthy they have used a lot of cosmetics in creams and other recipes so as essential oils they are natural they are pure but when we extract they are highly purified and natural they have not contain any synthetic compounds in the products that we use in Pakistan one of the famous countries is France they use all the synthetic they use esters and they use all the chemicals mostly synthetic these compounds the peels particularly are a key component of all essential oils the peels here are all essential oils and they are characterized so the family the class of compounds are all present so the distinctive character of essential oil can be attributed the functional group that is present in its three molecules esters ethers aldehydes lektone ketones these all are the functional groups which are present the compounds that are isolated from the essential oil if we look the methods how it can be extracted so there are so many methods are available if you can read any review article a large number of methods some are advanced methods that are trained to achieve or extract these essential oil so if I want to discuss here the two most important methods which are using from many years in past still it is famous in the laboratory we can isolate these essential oils so that is called hydro distillation this is one method what happen in hydro distillation you can take the material from where you want to extract the essential oil from flowers like a jasmine, like a rose and some other flowers if you want to extract from lavender you want to extract from our citrus family there is lemon hair orange hair red blood hair lots of species here you can take the material and just here you can see put the material inside the flask and you can add the water dip the material in the water and then you can give the heat and then evaporate it the water is evaporated it can also evaporate by essential oil from those peels and the glass materials and it can be condensed you can stir it over the biochemistry condensation process it can be condensed the condensation here in the price of water it can be separated by essential oil the density is very low so the water starts flowing down and the essential oil is evaporated you can extract by using this methods in the water and you can collect the essential oil that is present in the surface of the water so this is one of the simple methods soaking, heating and then you can volatile and condensation and at the last step you can separate and you can characterize by using the techniques and particularly the good one technique is GCMS you can characterize the compounds which can give you the idea about the components which we nature of contain by applying in practice essential oil there is another methods which is also used for the extraction of essential oil this is the steam distillation in this methods instead of dipping the sample in water you can produce the steam you can produce the steam and this steam can pass the material in which you want to extract the essential oil and that can be passed on to the condenser condenser always cool down the evaporated weapons and convert into liquid and here you can see is the oil is collected well and the down is the water you can remove the water and finally you can collect the essential oil in the steam distillation process in this process you cannot dip the material just you pass on the steam and it can extract the essential oil so if you look the modern uses of this particularly class of compounds that are extracted in the form of essential oil here you can see the uses of these essential oil in different actors it is involved in the cleaning because we have the anti-buffin activity it is also used for the flavor some they are good flavor you can use to obtain a very good flavor they are used in medical medicines insect repellent is very good if you use natural sources mosquito you can smell it for repellent you can use a perfume although it is very expensive but in photo class it can be used in perfumes and also it can be used in dentistry dentistry this is used in toothpaste in thiamol and many compounds they use essential oil they have good smell and help to clean the material form of the toothpaste so they are used in thiamol compound which is extracted from thiamol thiamol they are used to make these things like you see this one yes in detail many plants can be used and used for natural source and one of the most important is cosmetics they are used by essential oil for this cosmetics and the other soaps and some other sectors that are not mentioned here so if you look the classification of the food this is our function food so they are classified into 5 big groups the first one is known as conventional or basic food second one is fortified food you can see there is a market available and buy back there is a market and fortified and then andri's foods are genetically modified foods and the last one is enhanced food what are these different types of food and how they are classified if you look the examples are there small brief definitions so conventional foods are called basic foods they contain only nutrients or bioactive components they contain some nutrients and they have the bioactive components they are called the conventional foods or basic foods what is fortified food fortified food basically food with the nutrients have been added in addition to what have lost in the processing when processing is done when the oil is processed and if it is hydrogenated when the oil is heated during that high temperature during that processing when the oil is heated then the additional things add that is called fortified in this in vitamin A vitamin B this is the value of the oil in the process of loss in the synthetic sang they add that 45 to 45 because they have some additional components. So, what is enriched foods? Fruits to which new nutrients are components which are not normally present, normally found or added. Fruits to which many things are not present. So, to make them useful, we add a few things. What is enriched foods? Fruits to which new nutrients are not normally present, normally found or not. So, government should focus on the fact that to achieve iron deficiency, we enrich it and add iron or more or minus 7 and more quantity. Then, altered or radically modified foods, foods in which some potentially harmful existing components have been replaced with beneficial carbons. Some foods are like things which have a lot of health but are not useful. There should be a lot of health. So, there should be a lot of health for our body but we should add some other things to it. We should alter it. Genetically, we should go to the gene level and modify it in gene. For example, the seeds that are present in the body, which are seeds of health, sun flower, canola oil, there are very few seeds. When we extract the oil, they give 25-30% of oil. So, we should go to the gene level and enhance the oil. Then, what we need is polyunsaturated fatty acids. We should genetically modify it and enhance it. We should alter it. This is called the genetically modified or altered food that is contained by special nutrients, which is not normally present. The last one is an enhanced food that is changing raw commodities to hyperalcoholic compounds. So, there are raw commodities. There are less seasonings. They can be used in the food market. These are the bad types of foods that are enhanced foods. Here, we look at the examples of these five classes of the food. The basic food, which is contained oat, barley, wheat, bran, cereal, salt based product, green tea, seed, hemp, flax seed. These all are the basic foods. They contain the nutrients and health benefits. If you look at the certified food, like calcium certified milk and beverages. So, milk is available in the market these days. They have more calcium. Normally, calcium is less in milk. But, to enhance it more, it has more nutrition. In fact, the level of calcium is higher. In many countries, calcium deficiency can be used in high calcium water. Yogurt with probiotics. The probiotics are important. To enhance our flora intestines, we use probiotics because our intestines are flora. They are too healthy. When they are too healthy, they produce vitamin K. As well as, they cannot provide the attachment of the foreign particles. When foreign particles are attached, they will not be able to create a permanent basis. Binks with the herbal extract. Today, you see that in restaurants, the mint that is used in food, it contains the herbal extract. So, the bottle is not good for health. It is especially used for health. But, if you don't have it, you can use it for your health. In this way, folic acid and iron-45 breads in cereals, vitamin B-45, margarine, and iodized salt. These are all the examples of 45-2, which are nowadays commonly available in the market. Enriched food. Like vitamins and mineral-enriched products, like white bread, products containing added fiber, protein, and zinc biopets. So, these are the enriched foods. Enriched foods are intelligent. All of these are the enriched foods. All of these are the quality of the foods. All of these are the quality of the foods. All of these are the quality of the foods. This is the product with the added omega-3 fatty acid. Omega-3 fatty acid is an important fatty acid for our body. And it can not synthesize in our body. We must obtain them from a diet. So, omega-3 fatty acid is added. It contains ghee or whatever we use in it. Omega-3 fatty acid is added. It is enriched to quality. It is increased. Cation-enriched food juices. Then, iron-enriched breakfast cereal. And plant-steen-holes and steel-hole esters. Enriched margarine. These are all the examples of the enriched food. Enriched food, which are nowadays available in the market. Altered or genetically modified food. If a component is toxic, it can be removed. For example, if a component is allergic to a particular population, it can be modified to a certain level. And if a component is responsible to cause the allergy, it can be removed. So, in this way, most of the examples are from Muslim, where you can see the reduced fat and fat-3 products. Fats are then out. The problem with products is the increased fiber content. The fiber content will increase, and they are good for our stomach. In this way, sugar and salt are the free things to be made. So, the sugar will come to the things in which salt comes to them. These are all types of food that are genetically modified food. And it can be altered or knocked out by a specific gene, which is responsible to cause some specific things that are produced in a high quantity in any food. Enhanced food. Like a golden rice with enhanced beta-carotene contents. Enhanced production of vitamin E, because this vitamin E is act as anti-oxidants. So, it can be present in different steps in a high quantity. Lycopene enhanced in water, as it might be in a lycopene version, but we will enhance it more. Because they are good for the cure of fat, or prevention of cancer, or postage cancer, in particular. Highlycein, corn, foods and vegetables with enhanced contents of vitamin, carotene diet containing potatoes. So, these are all things that can be enhanced. The normal level and its level of enhancement of the food are good for the health. Here, you can see the typical mutasuticas. Nowadays, if you visit the market, particularly in advanced countries, in the pharmacies, there are shelves which have the mutasuticas and vitamin pills and they are obtained from the natural source. Mungal pills have the positive efficiency when the immune system is strong over. The foods will be protected from the diseases. Herbal supplements, oil gel caps, these are commonly available in the market. In the pharmacies, particularly in advanced countries, they are moving to the natural sources because synthetics always creates problems. In the past, there used to be a lot of chemistry in the case of synthetics using vitamin, but synthetics are not compatible with the body. So, now, again, we are shifting on natural sources. Natural sources are used for the food producers who are expensive. Advanced countries may have shelf life in the pharmacies when the synthetics are available in the natural sources. Those people who can easily afford so they can purchase natural organic stuffs and go and save for the health. Here, you can see the three probiotics, functional foods. You can see different yogurts, juice, different food stuffs are in the market available. They contain some more fiber, some more proteins like probiotics. Juice contains some additional benefits or additional components so you can purchase these from the market. Here, another example, you can see the juice which contains anti-oxidants in a high amount like biscuits or other stuffs which are more fiber and other small children with food stuffs and they also contain more fiber because fiber is good for our health. They are easily digest and good for the stomach. But the most important is the natural source. Always the natural source is the important. You can instead of focus to the processed material of food stuffs, you can use directly the vegetables which have good contents of the nutraceuticals. They have different components and they prevent you and also they are responsible. If it happens from a disease, you can use a high amount of anti-specific food and it can be cured. It can be able to cure your disease if it happens. So these are the few vegetable fruits you can use on daily basis in your food. Here you can see another strawberry is like a yogurt. It contains the probiotic food greens and it also contains the multivitamins. So these greens and these yogurts contain more probiotics and it also contains some additional vitamins. Here another company is a bunny call which has a claim that their product is responsible to remove the cholesterol because the heart disease is one of the top-leading diseases which cause the death in the human being. The highest rate is the heart disease. Heart cancer. Heart diseases are very common in people who have heart disease. The bunny call company they use the sterile sterols extracted from the plants sources which is more safer and they use in their cheese and in their peppers and they claim that their product is a layer of cholesterol higher in the cholesterol. So all these things are important but there are certain claims that come from the pharmaceuticals and the health claims. What are these claims? Early development and growth. If you use these butycyticals they do the good development and proper growth. Revolution of basic metabolic process defense against oxidative stress and certain cancers. Cardiovascular and gastrointestinal improvements improve mental health and new response and enhancing effects and also the optimal defense against infectious diseases. So these are the claims that comes from the pharmaceuticals. Those pharmacies, there is industries who are selling the pharmaceuticals. They have come in these claims that the pharmaceuticals have these properties. So regulatory bodies is important in every country. The administration in Pakistan will have less regulatory bodies in different sectors and also the issue in the functioning of the pharmaceuticals still will have problems but in advanced countries regulatory bodies, if you can expect something, you can prepare some food stuff you can get a licensing you can get some testing from us in different regulatory bodies you can see from Brazil, from Canada, from China they have their regulatory bodies they can check your claim however your pharmaceutical or functioning foods is performing specific functions after checking to allow you you can sell the things in the market in foreign countries have regulatory bodies to check the quality of the pharmaceuticals and actually foods. Now the important thing is the market over here. In Pakistan, there is no statistical guidelines available but how much things or how much money we generate by using this pharmaceutical or functioning foods but here you can see some advanced countries are available this is a very good market because in the coming years people are focusing to use the functioning foods so in each year the production in terms of finance this is increasing here you can see its claim that its business was in 2017 299 billions US dollar business of these industries now it is increasing and they are projected they are hopeful that it will reach 400 approximately 450 billion US dollars by the end of this year so we have a lot of opportunity to develop some food stuffs and sold in the market this is a good chance that because people now are convinced the nutritional and functioning foods are very good for our health so we are purchasing these stuffs and obviously we are from the other big markets we have good gaps to as each sector is the most important matter so we can develop some good food stuffs we are adding some new nutrients or useful nutrients and we can do so here you can see from China from Europe or North America Asia Pacific you can see these are already available in North America billion dollars by using these nutrients so now we come to the problems because it looks like a simple in fact it is not simple it is challenging and many problems are still is to develop these nutritional and functioning foods the first if you look at delivery many biologists are unstable there is a lot of issue here which we isolate we are unstable chemically instability in oxidation of acne we can't go back here physiologically and physically stability oxygen and moisture these two are responsible to damage these bioactives so we have still problems we need to overcome these things that we can protect our bioactor compound when we are isolated from infiltration so many bioactives in fact negatively are organo-laptic of fruits and beverages some are very useful but in fact they are organo-laptics this is our language and we don't accept it many taste is chain now what we do is very good thing because it is tasty for example in medicine it is called white blood in the name of white blood it is so bitter that it doesn't even drink when you don't want to drink it it doesn't drink organo-laptic is important when you alter when you try to make complete food then it starts to change taste so it is difficult to accept the taste this is another issue that when you cut with muscles then taste changes taste, odor, food, texture, mouth feel texture changes feeling in mouth after eating the taste gets spoiled so as much as it is useful it will be very difficult we will not eat food for our mouth many existing delivery systems are unsuitable for wide food, beverages and applications it is expensive material it is very important to process it it is expensive it is not available it will be successful beverages will be successful in different food production then development of suitable food vehicles for target delivery of biotechers specific vehicles will be transferred to biotech compound delivery of biotech compounds at desired levels then acceptability stability and bioavailability bioavailability is also important if we take it and if it is not available in blood then it will be useful so bioavailability is checked that when we are taking components either they are present or they are not if they are present if bioavailability is not available if it is not digested if it is not achieved at the level then our target will not be achieved then anticipate demand for personalized nutrition and the potential role of functional ingredients for each person for each person ensure stability of functional food ingredients during manufacturing target delivery then ensure quality of foods if it is not achieved then your objectives will not be achieved then need to define the bioavailability of functional ingredients for each nation how much level should be available how useful and how high level will be now iron is good calcium is good but if we take it in high quantity then that will create problem calcium deposit is in our kidney and they will cause the stud calcium sulfate carbonate then establish dietary referencing take for a wide range of nutrients they establish dietary here how useful how useful and how useful challenges are still we isolate we characterize we convert feed and make nutrition then other related issues public health education now clean educate them that these nutrition natural source are good investment to bridge research gap lack of knowledge in the intellectual property protection then insufficient coordination of research activity of your food karma loading or chemistry loading until we work together definitely the development of food nutrition food for pharmacy synthetic compounds chemistry is not convinced chemistry because they are doing synthetic work they are not convinced they do not believe in this because synthetic is not natural the biggest claim is synthetic is on a large scale production source have a lot of work they can provide you see chemistry is not convinced there is a difference between synthetic and natural source then limited financial resources this is another issue which can not generate nutrition guys and a small number of large companies dominate and shape market they have not done marketing then small companies do not run small companies we do not have a multinational company that is not so wrong so that family does not get cured then they are discouraged that you do not use multinational company we are scientifically trained personal people are trained and need for search of potential emerging market this is another issue to see our potential which components we need which foods we need we can do we can do we can do science is also responsible to generate money scientist job is responsible to generate money in science we spend 1 rupee 10 rupees we spend 10 rupees this is our issue in China economy is going ahead we are making things cheap we want to make money 100 rupees 1 rupee industries business plan you are just talking or in fact you will be able to do something or cheaper view so if we conclude our talk function food containing physical active components either from plant source from animal source and it can improve health and possible reduce the risk of diseases cheap foods industry and resource intensive both in terms of financial resources and the time required for basic research technology development and commercialization to use all the aspects that are commercialized products we are promising technology such as metagenomics can be used for development of food or target population population groups with defined disease such as allergy, diabetes obesity and cardiovascular disease genetically modified foods and sugar salt fats patients cardiovascular patients we want to be products cost again then the last one conclusion of the talk is all about the process unique nutrition and health characteristics functional foods still could not complete the conventional food for the market share and shelf space in the retail stores still you have a famous menu because the menu does not care about other people who are uneducated they do not care what they are eating so education is also necessary but still we have the web if we develop some good food stuff we can sell in the market so thank you for your patience thank you I will give you a presentation I will give it to you I will give it to you I will give it to you thank you thank you see this question most people who use this question they use it when they use it they do not know what is this the problem is because it is not available the problem is it is a mixture of things and they do not care that is why the cancer is very common actually according to the people who do not know what are these what are these if they do not know what is this if they are eating high salt they should know that they have to care if they have a heart then they are eating meat they are using fat they are using ghee now things are changing people are becoming convinced people in villages they are using natural sources to remember but there are many things that are available on the market so people take it there is a contamination and it is used for synthetic things for example when we eat sweet rice they use 6-7 colors and the synthetic is very good now they use natural colors from jamans mangoes but they are expensive they use 1000 rupees or 1500 rupees that is not available that is why people do not have money but if they want to buy they sell it they sell it but if they want to buy they sell it they sell it they sell it but if they do not buy they sell it so people have to educate that some of our old chemistry has been used for synthetic things now synthetic things are being declined there are many problems cancer has increased every fourth person in the world is suffering from cancer every fourth person and we do not have any care for example I saw a guy selling chicken pieces so he is an ampoule from agriculture he is a producer he sells ampoule he has a job now when they release a press when they press they release 20 pages of the press and they are black they are very good they sell it they sell it they sell it and the black pieces they sell it so 20 pages of the press and in the market we buy it we buy it only once maximum 2 times that is very bad like our country is very bad we buy it and buy it in other places and then in the cities it is a family business if you are working in other places if I am doing a quite small business I will give 1,000 the plastic packet it has a sort of bomb ring we have a part of the plastic we keep the plastic the buildup of the plastic it is 484 430 and the black pieces are used the water is removed it's not good for health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. It's a very bad impact on our health. Thank you so much. Thank you so much, sir. It was very knowledgeable and very interesting session with you. And ladies and gentlemen, this brings us to end the first session of the day. Let's take lunch break and resume the session at 2pm. It will be 30 minutes break now. Hi, Marta. I'm muted myself, sorry. Here is lunch break and we were a bit late in our first session. That's why people are coming back to auditorium. It may take 4-5 minutes, so we will resume at that time. Okay. Let me know when you're ready. Okay. This is my university auditorium. Lovely. How many people are we expecting in there? Sorry. How many people will you be expecting? How many students are on site? Most of the majority of the participants are faculty members from our university and from other universities. Oh, I see. But they are mostly Lahore based. I think the number is around 35-40. Because I can see 26 online as well? Yeah, it may increase in a few minutes because it was lunch break, so people will join us again. So I think 40, about 40 online and 40 in person here. Okay. And I realize it's a sort of mixture of talks, something about education, but also science. Looking at the schedule. Today they just had genomics lecture, is that right? Yeah, it was a mixture. Okay. Thank you. There is Ms. Samar. She is hosting this event. Hi, Dr. Marta. How are you? Hello. I'm good. Thank you. It's early morning here. Well, not early, but 9 o'clock. So early start in a way in comparison to you. We are just starting in one or two minutes. That's okay. So what city are you actually based? Because it's a virtual one. So I suppose people can join from wherever they are, but the auditorium, where is it physically? We are actually in Lahore, Pakistan, our head office, Virtual University of Pakistan. And the participants are also from Virtual University and other universities. Okay. And some participants, those are from outstation, they are on Zoom online. Yes. Ladies and gentlemen, welcome back. We will now resume our second session of the day. The speaker of this session is Dr. Marta Fatima. She is working as lecturer in Cardiovascular Science, Cardiology Research Center, Molecular and Clinical Science Research Institute, St. George University of London. Before that, she was serving as Senior Research Associate at Centre for Heart Muscle Disease Institute of Cardiovascular Science, University College London. Dr. Fatima is reviewed for numerous generals and a member of Genomics England Cardiovascular Domain of the OneLag Genomes Project. She is finalist of the Young Investigator Award Competition, an active member of the ICS Research and Academic Networking Group. Today she will deliver her talk on genomics of familial hypercholestrolomia from diagnosis to management. Welcome Dr. Fatima on behalf of Virtual University of Pakistan. Please proceed for your presentation. Thank you very much for this kind invitation and an introduction. It's a pleasure to join your event and to see many attendants in the audience and online. So yes, it's early morning here and I'm kind of full of energy but I suppose you just had your lunch break so you might be in a slightly different state. I hope I won't be presenting too much of heart science. I wanted to demonstrate to you based on the disease familial hypercholestrolomia, how you can utilize genomics, how you can improve patients identification, treatment and most of all prevent cardiovascular disease. Let me just find my slides so that I can share my screen with you. Can you see that? Can I see my screen? Yes, this is Dr. Fatima. I'll do full screen. Yes, so I am a lecturer at St. George's University but I still have close links with UCL where I did my PhD and two postdoc positions. Tomorrow I believe you will have a presentation from my ex-PhD supervisor, Professor Steve Hanfries. So we really enjoyed working together and we still continue some work on familial hypercholestrolomia. I will call familial hypercholestrolomia as FH, which is a little bit easier to pronounce. Today I will talk to you about the clinical characteristics of the disease. It is a disease of high LDL cholesterol and so we will cover the basics of that pathway, how that cholesterol is metabolized. FH is a genetic disease so we will cover the genetic causes of it and look at the genetic epidemiology of FH. So how common it is, what mutation causes all around the world. We will look at the diagnosis from clinical to genetic diagnosis and screening approaches for FH. And in the second part of my lecture I want to show you something more on genomics. So comparing monogenic versus polygenic causes of high cholesterol and how you can utilize genomics knowledge to identify novel causes of FH. And from that how you can go to novel treatment as well. So it will be genomics heavy, I don't know what exactly you just had on your previous lecture but I hope this is not going to be too much and perhaps you can relate. This would be a good example for you how you can use your previous knowledge. Yes, so hopefully that will be, we will be able to cover that all today. So what is familial hypercholestrolomia? What are the clinical characteristics? Well, it says a lot in its name. It is a disease that affects families and hyper part of the name means too much cholesterol and emia is from a Greek word that means in blood. So in its name we can understand that it is a disease that affects families due to high cholesterol levels in the blood. And indeed if we're talking about familial disease, I wanted to bring you an example of a family that is affected by FH. I didn't want to use the real picture so this is just a sketch of a family, a young family. We've got mum, Jeanette, who is 33 year old, dad, Mike, 35 year old, and they've got two sons. There is Daniel, who just turned five, and a toddler, Matthew, who is one year old. And so they are living busy family, young family life, both working parents, and suddenly at the age of 35, Mike unfortunately suffers from myocardial infarction, so an heart attack. And obviously this is a tragic news for a young family. It's not only a health issue for Mike, but also puts loads of pressure and stress on the family. And therefore I want to just highlight that as a familial disease, even if a person is not affected, there will still be affected in other ways, such as stress or that they will need to start looking after a member of their family. So one of the first things that doctors do when they treat, when they diagnose patients with myocardial infarction is they look at their cholesterol levels. And specifically LDL cholesterol, this is the bad cholesterol. So they did that for the whole family in Mike's case, because they were already, I think, suspecting familial hypercholesterolemia as the heart attack occurred very early on. And indeed Mike's cholesterol levels, LDL cholesterol was 6.8 millimore per liter. If you compare him to the cholesterol levels of his wife, it is much higher. And they both have similar diets, so you kind of think you could exclude any dietary issues here. They also, the doctors decided to look at the children and I will expand on that later, why would they be looking into children. But it would be mainly because of you might be able to prevent such events in the future. So let's just bear in mind that Daniel has got LDL cholesterol slightly lower than his mom. And he's a five year old, whereas Matthew has cholesterol level of 2.5 millimore per liter. So we'll come back to that later. So clinical characteristics of a phage includes the fact that it is an autosomal dominant disease. What that means is that 50% of your first degree relatives would inherit a mutation. So your children or your parents would also be affected. Therefore, when talking about a phage it is very important to analyze the family history of either early myocardial infarction or very high cholesterol levels. Having 50% of your first degree relatives really strongly suggest that this is a ph that there is something that is strongly inherited. It is a disease of high LDL cholesterol, so the bad cholesterol and we will talk about LDL a little bit more later. But that cholesterol levels is people with a phage. They are exposed to that high cholesterol levels really from birth and although perhaps in children this isn't visible. It's kind of a silent risk factor. The cholesterol level accumulates over time with age that leads to a very high risk of premature heart attack. And the FH foundation estimated that one in five heart attacks under the age of 45 are due to familial hypercholesterolemia. So pretty serious issue. Let's think about Mike again and think how or what caused this heart attack in his case. So to talk about heart attack, we always think of cardiovascular systems. So here is a picture of an artery, a healthy artery. We've got the lumen, the arterial wall. And so we know that Mike had hyperepidemia or high cholesterol levels. So clearly would you have high cholesterol level over time that leads to a buildup of that fatty substance in the arterial wall. A process called atherosclerosis. There is a sort of overtime plug that forms. There is an inflammation going on. So loads of bad things are happening, but this is still kind of okay until the moment when this plaque ruptures. And when it ruptures, it will form a clot. And the clot is really the very dangerous part of that atherosclerosis and a plaque formation. The clot may block an artery and that will prevent obviously the blood flow, oxygen flow to organs. And if it happens near heart, we will have muscle damage. And that is the myocardial infarction. So really, if you think about the cause in terms of Mike's case, we would probably need to start thinking about why he's got high cholesterol rather than looking at the downstream events. So we want to go back to this point why there is high LDL cholesterol in this person and could this then downstream events be prevented. So I think to answer the question why a person may have high cholesterol, we would need to look at how is actually cholesterol cleared in our body. So at least here in Western Europe, we get loads of bad press for cholesterol. It's kind of, we know we should not be eating fatty diet and also our environment is full of fatty food. So the cholesterol itself, LDL cholesterol gets very bad press in a way. But it is a very important component in our body. So we must remember that every single cell in our bodies is composed needs cholesterol for its membranes. Hormones are synthesized from cholesterol by bile acids, as well as it's important precursor for vitamin D which plays many important roles. And the main organ in our body that does the job of metabolizing cholesterol is the liver. So really about 20% of the cholesterol that we have in our body actually comes from the diet. And we as humans developed the process of synthesizing our own cholesterol in the body. So clearly that highlights the fact that it is a very important component and humans simply cannot leave without it. We need it. And as I said, we have a process in which we can synthesize the cholesterol ourselves. And that happens in the liver. So LDL cholesterol is the main type of cholesterol in human body. Of course, you might have heard of HDL cholesterol. This is the good cholesterol. So VLDL cholesterol, very low density lipoprotein. Treglycerides, this is the dietary cholesterol. So there are several different types, but a majority of cholesterol in human body is transported in the form of LDL cholesterol. So it's a particle. It's kind of a packed particle with loads of cholesterol inside and it is wrapped around by a protein called apoB. So now if you imagine that it is a fatty substance, so if you drop a little bit of oil in a glass of water, you will see that it sort of floats on the surface of the water. It will not easily dissolve in the queen's solution. So if you think that cholesterol needs to be transported in circulation, in blood, and blood is mainly made of water, this cholesterol needs to be made slightly more hydrophilic. So it needs to like the water. And that's the role of apoB. ApoB as a large protein wraps around that fatty substance, that cholesterol, and makes it easier to be transported in blood. And liver and many other types of cells, but mainly liver has or expresses LDL receptors. And these are sort of like a seven-fingered hands that are sitting on the surface of a cell and they would wait. Their function is to bind the cholesterol from circulation. And so it is the apoB part of the cholesterol. So I've marked it here like this green color. This is the apoB wrapping around the cholesterol. It is the apoB that binds the LDL receptor and that triggers endocytosis. So this cholesterol goes inside the cell where it is metabolized. And next, the LDL receptors can be recycled back to the surface of the cell or can be degraded. The process of the recycling and degradation of LDL receptors is controlled by an enzyme called PCSK9. So hopefully you can see these three major players here in that pathway. We have the LDL receptors. We have apoB that binds to LDL receptors. And then we have PCSK9 that degrades LDL receptors. And that work was mainly done by Professors Brown and Goldstein who received a Nobel Prize back in 1985 for their work on cholesterol metabolism. They were the first FH mutations actually came from their lab as well, LDLR mutations. Even when I look at this picture here, I think they drone here actually sort of a symbolic LDL receptor. So this is kind of like that hand that is sticking out waiting to bind apoB. So if you're more interested into that, you could search for their names and this is the sort of pioneering work on LDL receptor and LDL cholesterol. So FH is due to the defective clearance of plasma LDL cholesterol. And that obviously then leads to high cholesterol in the circulation. So thinking about the previous slide, the cartoon, you could conclude that perhaps this faulty clearance of cholesterol could be because of dysfunctional LDL receptors that cannot bind the cholesterol. Perhaps the binding between the apoB and LDL receptors is insufficient and that also leads to increased circulation, concentration of LDL cholesterol. Or maybe there is an enhanced degradation of the LDL receptors because of the PCSK9 function. And indeed, mutations in these three players in LDL receptor pathway are known to cause FH. So we've got mutations in LDL receptor gene that lead to either lack of receptors or dysfunctional receptors. We know that they cause familial hyperchrystalline. Mutations in the apoB protein decrease affinity of the apoB to the receptor and they also lead to FH. And mutations, these are gain of function mutations, so they improve the function of PCSK9. And so PCSK9 starts degrading LDL receptors at a higher rate and that leads to less receptors being available on the cell surface to bind LDL cholesterol. So these are the three important players in FH. There are novel genes coming up and I will cover a little bit more about that later. Probably most convincing recent mutation was reported in apoE gene. There is a one single variant. It's a deletion of lusine at a position 167 that has been suggested to lead to FH. There was a nice family co-segregation study. But looking at our population data, recent ones, we think this variant might be slightly milder than the known FH mutations. There is another form of FH. It's a recessive familial hyperchrystalline, which I will not be covering today, but I want you to be aware of it. This is due to mutations in LDL are adapted protein one and that protein responsible for formation of those coated pits where which are involved in the endocytosis of LDL receptors. But this is a recessive FH, so slightly different inheritance pattern, and we will not be talking much about it today. So if we look at the genetic causes overall, about 80 to 90% of all FH mutations are in the LDL receptor gene. And so this is the most common cause of FH. Applebee, it's a very large gene, mostly understudied, I think now with next generation 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 again quite a substantial proportion of the cases. 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. If you are a homozygot, you clearly don't have the functional LDL receptor because you inherited fault, you run from mom and from dad. 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 those individuals are healthy, living normal life. 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.