#133 – Manolis Kellis: Biology of Disease

The following is a conversation with Manoa's Kellis, his third time on the podcast. He is a professor at MIT and head of the MIT Computational Biology group. This time we went deep on the science, biology and genetics.

So this is a bit of an experiment. Manos went back and forth between the basics of biology to the latest state of the art and the research. He's a master at this. So I just sit back and enjoy the ride. This conversation happened at 7 a.m. So it's yet another podcast episode after an all-nighter for me. And once again, since the universe has a sense of humor,

This one was a tough one for my brain to keep up, but I did my best and I never shy away from a good challenge. Quick mention of each sponsor, followed by some thoughts related to the episode. First is SEMrush, the most advanced SEO optimization tool I've ever come across. I don't like looking at numbers, but someone probably should. It helps you make good decisions.

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Please check out the sponsors in the description to get a discount and to support this podcast. As a side note, let me say that biology in the brain and in the various systems of the body, fill me with awe. Every time I think about how such a chaotic mess coming from its humble origins in the ocean was able to achieve such incredibly complex and robust mechanisms of life that survived despite all the forces of nature that want to destroy it.

It is so unlike the computing systems we humans have engineered that it makes me feel that in order to create artificial general intelligence and artificial consciousness, we may have to completely rethink how we engineer computational systems.

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Okay, this show is also sponsored by 8Sleep and it's Pod Pro mattress that you can check out at 8sleep.com slash Lex to get $200 off. It controls temperature within app. It is packed with sensors and can cool down to as low as 55 degrees on each side of the bed separately. And it totally has been a game changer for me. I don't particularly like fancy material possessions as you may or may not know.

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Check it out at betterhelp.com slash Lex. And now here's my conversation with Manolis Callis.

So your group at MIT is trying to understand the molecular basis of human disease. What are some of the biggest challenges in your view? Don't get me started. I mean, understanding human disease is the most complex challenge in modern science. So because human disease is as complex as the human genome, it is as complex as the human brain.

And it is in many ways even more complex because the more we understand disease complexity, the more we start understanding genome complexity and epigenome complexity and brain circuitry complexity and immune system complexity and cancer complexity and so on and so forth. So traditionally,

Human disease was following basic biology. You would basically understand basic biology in model organisms like, you know, mouse and fly and yeast. You would understand sort of mammalian biology and animal biology and eukaryotic biology in sort of progressive layers of complexity, getting closer to human phylogenetically. And you would do perturbation experiments in those species.

to see if I knock out a gene, what happens? And based on the knocking out of these genes, you would basically then have a way to drive human biology because you would sort of understand the functions of these genes. And then if you find that a human gene locus, something that you've mapped from human genetics to that gene is related to a particular human disease, you'd say, ha, now I know the function of the gene from the model organisms.

I can now go and understand the function of that gene in human.

But this is all changing. This is dramatically changed. So that was the old way of doing basic biology. You would start with the animal models, the eukaryotic models, the mammalian models, and then you would go to human. Human genetics has been so transformed in the last decade or two that human genetics is now actually driving the basic biology. There is more genetic mutation information in the human genome than there will ever be in any other species.

What do you mean by mutation information? So perturbations is how you understand systems. So an engineer builds systems and then they know how they work from the inside out. A scientist studies systems through perturbations.

You basically say, if I poke that balloon, what's going to happen? And I'm going to film it in super high resolution, understand, I don't know, air dynamics or fluid dynamics, if it's filled with water, et cetera. So you can then make experimentation by perturbation. And then the scientific process is sort of building models that

best fit the data, designing you experiments that best test your models and challenge your models and so on and so forth. That's the same thing with science. Basically, if you're trying to understand biological science, you basically want to do perturbations that then drive the models. So how do these perturbations allow you to understand disease?

So if if you know that a gene is related to disease you don't want to just know that it's related to the disease you want to know what is the disease mechanism because you want to go and intervene. So the way that I like to describe it is that traditionally.

epidemiology, which is basically the study of disease, you know, sort of the observational study of disease, has been about correlating one thing with another thing. So if you have a lot of people with liver disease or also alcoholics, you might say, well, maybe the alcoholism is driving the liver disease, or maybe those who have liver disease self-medicate with alcohol, so that the connection could be either way.

with genetic epidemiology, it's about correlating changes in genome with phenotypic differences. And then you know the direction of causality. So if you know that a particular gene is related to the disease, you can basically say, okay, perturbing that gene in mouse causes the mice to have X phenotype.

So perturbing that gene in human causes the humans to have the disease. So I can now figure out what are the detailed molecular phenotypes.

in the human that are related to that organismal phenotype in the disease. So it's all about understanding disease mechanism, understanding what are the pathways, what are the tissues, what are the processes that are associated with the disease so that we know how to intervene. You can then prescribe particular medications that also alter these processes. You can prescribe lifestyle changes that also affect these processes and so on and so forth.

That's such a beautiful puzzle to try to solve what kind of perturbations eventually have this ripple effect that leads to a disease across the population. And then you study that for animals, mice, first and then see how that might possibly connect to humans. How hard is that puzzle of trying to figure out how little perturbations might lead to in a stable way to a disease?

In animals, we make the puzzle simpler because we perturb one gene at a time. That's the beauty of the power of animal models. You can basically decouple the perturbations. You only do one perturbation and you only do strong perturbations at a time. In human, the puzzle is incredibly complex.

Because, obviously, you don't do human experimentation, you wait for natural selection and natural genetic variation to basically do its own experiments, which it has been doing for hundreds and thousands of years in the human population and for hundreds of thousands of years across the history leading to the human population.

So you basically take this natural genetic variation that we all carry within us. Every one of us carries six million perturbations. So I've done six million experiments on you, six million experiments on me, six million experiments on every one of seven billion people on the planet. What's the six million correspond to? Six million unique genetic variants that are segregating in the human population.

Every one of us carries millions of polymorphic sites. Poly, many morph forms. Polymorphic means many forms, variants. That basically means that every one of us has single nucleotide alterations that we have inherited from a moment from that, that basically can be thought of as tiny little perturbations.

Most of them don't do anything, but some of them lead to all of the phenotypic differences that we see between us. The reason why two twins are identical is because these variants completely determine the way that I'm going to look at exactly 93 years of age. How happy are you with this kind of data set? Is it large enough of the human population of Earth? Is that too big, too small?

Yeah, so is it large enough is a power analysis question. And in every one of our grants, we do a power analysis based on what is the effect size that I would like to detect and what is the natural variation.

in the two forms. So every time you do a perturbation, you're asking, I'm changing form A into form B. Form A has some natural phenotypic variation around it, and form B has some natural phenotypic variation around it. If those variances are large and the differences between the mean of A and the mean of B are small, then you have very little power. The further the means go apart, that's the effect size, the more power you have, and the smaller the standard deviation,

the more power you have. So basically, when you're asking, is that sufficiently large? Certainly not for everything, but we already have enough power for many of the stronger effects in the more tight distributions. So that's a hopeful message that there exists parts of the genome that have a strong effect that has a small variance.

That's exactly right. Unfortunately, those perturbations are the basis of disease in many cases. So it's not a hopeful message. Sometimes it's a terrible message. It's basically, well, some people are sick. But if we can figure out what are these contributors to sickness, we can then help make them better and help many other people better who don't carry that exact mutation.

but who carry mutations on the same pathways. And that's what we like to call the allelic series of a gene. You basically have many perturbations.

of the same gene in different people, each with a different frequency in the human population and each with a different effect on the individual vicarism. So you said in the past there would be these small experiments on perturbations and animal models, what does this puzzle solving process look like today?

So we basically have something like 7 billion people on the planet, and every one of them carries something like 6 million mutations. You basically have an enormous matrix of genotype by phenotype, by systematically measuring the phenotype of these individuals. And the traditional way of measuring this phenotype has been to look at one trait at a time.

You would gather families and you would sort of paint the pedigrees of a strong effect, what we'd like to call Mendelian mutation. So a mutation that gets transmitted in a dominant or a recessive, but strong effect form, where basically one locus plays a very big role in that disease.

You can then look at carriers versus non-carriers in one family, carriers versus non-carriers in another family, and do that for hundreds, sometimes thousands of families, and then trace these inheritance patterns, and then figure out what is the gene that plays that role. Is this the matrix that you're showing in talks or lectures?

So that matrix is the input to the stuff that I saw in talks. So basically that matrix has traditionally been strong effect genes. What the matrix looks like now is instead of pedigrees, instead of families, you basically have

thousands and sometimes hundreds of thousands of unrelated individuals, each with all of their genetic variants and each with their phenotype, for example, height or lipids or whether they're sick or not for a particular trait.

That has been the modern view, instead of going to families, going to unrelated individuals with one phenotype at a time. And what we're doing now, as we're maturing in all of these sciences, is that we're doing this in the context of large medical systems or enormous cohorts that are very well phenotyped across hundreds of phenotypes, sometimes with our complete electronic health record.

So you can now start relating, not just one gene, segregating one family, not just thousands of variants segregating with one phenotype, but now you can do millions of variants versus hundreds of phenotypes. And as a computer scientist, I mean, deconvolving that matrix, partitioning it into the layers of biology that are associated with every one of these elements is a dream come true. It's like the world's greatest puzzle.

And you can now solve that puzzle by throwing in more and more knowledge about the function of different genomic regions and how these functions are changed across tissues and in the context of disease. And that's what my group and many other groups are doing. We're trying to systematically relate this genetic variation with molecular variation at the expression level of the genes.

at the epigenomic level of the gene regulatory circuitry, and at the cellular level of what are the functions that are happening in those cells, at the single cell level, using single cell profiling, and then relate all that vast amount of knowledge

Computationally, with the thousands of traits that each of these of thousands of variants are perturbing.

And then it's not just like a puzzle of perturbation and disease. It's perturbation then affected the cellular level and at an organ level.

How do you disassemble this into like what your group is working on? You're basically taking a bunch of the heart problems in the space. How do you break apart a difficult disease and break it apart into problems that you, into puzzles that you can now start solving? So there's a struggle here. Computer scientists love heart puzzles and they're like, oh, I want to build a method that just deconvolve the whole thing computationally.

That's very tempting and it's very appealing, but biologists just like to decouple that complexity experimentally, to just peel off layers of complexity experimentally. That's what many of these modern tools that my group and others have both developed and used. The fact that we can now figure out tricks for peeling off these layers of complexity by testing one cell type at a time.

or by testing one cell at a time. And you could basically say, what is the effect of this genetic variant associated with Alzheimer's on human brain? Human brain sounds like, oh, it's an organ. Of course, just go one organ at a time. But human brain has, of course, dozens of different brain regions. And within each of these brain regions, dozens of different cell types.

And every single type of neuron, every single type of glial cell between astrocytes oligodendrocytes, microglia, between all of the neural cells and the vascular cells and the immune cells that are co-inhabiting the brain between the different types of excitatory and inhibitory neurons that are interacting with each other between different layers of neurons in the cortical layers.

Every single one of these has a different type of function to play in cognition, in interaction with the environment, in maintenance of the brain, in energetic needs, in feeding the brain with blood, with oxygen, in clearing out the debris,

That are resulting from the super high energy production of cognition in humans so all of these things are basically.

potentially deconvolvable computationally, but experimentally, you can just do single cell profiling of dozens of regions of the brain across hundreds of individuals, across millions of cells, and then now you have pieces of the puzzle that you can then put back together to understand that complexity. I mean, first of all, I mean, the cells in the human brain are the most, okay, maybe I'm romanticizing it, but cognition seems to be very complicated.

So separating into the function, breaking Alzheimer's down to the cellular level seems very challenging. Is that basically you're trying to find a way that

some perturbation and genome results in some obvious major dysfunction in the cell. You're trying to find something like that. Exactly. So what does human genetics do? Human genetics basically looks at the whole path from genetic variation all the way to disease.

So human genetics has basically taken thousands of Alzheimer's cases and thousands of controls matched for age, for sex, for environmental backgrounds and so on and so forth. And then looked at that map where you're asking what are the individual genetic perturbations and how are they related to all the way to Alzheimer's disease.

And that has actually been quite successful. So we now have, you know, more than 27 different low side. These are genomic regions that are associated with Alzheimer's.

at this end-to-end level. But the moment you sort of break up that very long path into smaller levels, you can basically say from genetics, what are the epigenomic alterations at the level of gene regulatory elements, where that genetic variant perturbs the control region nearby. That effect is much larger.

You mean much larger in terms of as down the line impact or it's much larger in terms of the measurable effect, this A versus B variance is actually so much cleanly defined when you go to the shorter branches because for one genetic variant to affect Alzheimer's, that's a very long path. That basically means that in the context of millions of these six million varies that every one of us carries, that one single nucleotide has a detectable effect.

all the way to the end. I mean, it's just mind-boggling that that's even possible. But indeed, there are such effects. So the hope is, or the most scientifically speaking, the most effective place where to detect the alteration that results in disease is earlier on in the pipeline as early as possible. It's a trade-off.

If you go very early on in the pipeline, now each of these epigenomic alterations, for example, this enhancer control region is active, maybe 50% less, which is a dramatic effect. Now you can ask, well, how much does changing one regulatory region in the genome in one cell type change disease? Well, that path is now long. So if you instead look at expression,

The path between genetic variation and the expression of one gene goes through many enhancer regions, and therefore it's a subtler effect at the gene level, but then now you're closer because one gene is acting in the context of only 20,000 other genes, as opposed to one enhancer acting in the context of 2 million other enhancers. So you basically now have genetic epigenomic, the circuitry, transcriptomic, the gene expression level,

And then cellular, where you can basically say, I can measure various properties of those cells. What is the calcium influx rate when I have this genetic variation? What is the synaptic density? What is the electric impulse conductivity? And so on and so forth. So you can measure things along these

path to disease. And you can also measure endophenotypes. You can basically measure your brain activity. You can do imaging in the brain. You can basically measure, I don't know, the heart rate, the pulse, the lipids, the amount of blood secreted and so on and so forth. And then through all of that, you can basically get at the path to causality, the path to disease.

And is there something beyond cellular? So you mentioned lifestyle interventions or changes as a way to, um, or like be able to prescribe changes in lifestyle. Like what, what about organs? What about like the function of the body as a whole?

Yeah, absolutely. So basically, when you go to your doctor, they always measure your pulse, they always measure your height, they always measure your weight, your BMI, so basically these are just very basic variables. But with digital devices nowadays, you can start measuring hundreds of variables for every individual. You can basically also phenotype cognitively through tests.

Alzheimer's patients. There are cognitive tests that you can manage, that you typically do for cognitive decline, these mini-mental observations that you have specific questions to. You can think of enlarging the set of cognitive tests.

So in the mouse, for example, you do experiments for how do they get out of mazes, how do they find food, whether they recall a fear, whether they shake in a new environment, and so on and so forth. In the human, you can have much, much richer phenotypes, where you can basically say, not just imaging at the organ level, and all kinds of other activities at the organ level, but you can also do at the organism level, you can do behavioral tests,

And how did they do on empathy? How did they do on memory? How did they do on long-term memories versus short-term memory? And so on and so forth. I love how you're calling that phenotype. I guess it is. It is. But your behavior patterns that might change over a period of a life, your ability to remember things, your ability to be empathetic or emotionally, your intelligence, perhaps even. Yeah, but intelligence has hundreds of variables.

You can be your math intelligence, your literary intelligence, your puzzle-solving intelligence, your logic. It could be like hundreds of things. We're able to measure that better and better. All that could be connected to the entire pipeline. We used to think of each of these as a single variable like intelligence. That's ridiculous. It's basically dozens of different genes that are controlling

every single variable you can basically think of, you know, imagine us in a video game where every one of us has measures of, you know, strength, stamina, you know, energy left and so forth. But you could click on each of those like five bars that are just the main bars and each of those will just give you then hundreds of bars.

And you can basically say, okay, great for my machine learning task. I want someone who, I'm a human who has these particular forms of intelligence. I require now these 20 different things. And then you can combine those things and then relate them to, of course, performance in a particular task. But you can also relate them to genetic variation that might be affecting different parts of the brain.

For example, your frontal cortex versus your temporal cortex versus your visual cortex and so forth. Genetic variation that affects expression of genes in different parts of your brain can basically affect your music ability, your auditory ability, your smell. Just dozens of different phenotypes can be broken down into hundreds of cognitive variables and then relate each of those to thousands of genes that are associated with them.

So somebody who loves RPGs or playing games, there's too few variables that we can control. So I'm excited if we're in fact living in a simulation. This is a video game. I'm excited by the quality of the video game. The game designer did a hell of a good job. So we're impressed.

Oh, I don't know. The sunset last night was a little unrealistic. The graphics. Exactly. To zoom back out, we've been talking about the genetic origins of diseases, but I think it's fascinating to talk about what are the most important diseases to understand, and especially as it connects to the things that you're working on.

So it's very difficult to think about important diseases to understand. There's many metrics of importance. One is lifestyle impact. And if you look at COVID, the impact on lifestyle has been enormous. So understanding COVID is important because it has impacted the well-being in terms of ability to have a job, ability to have an apartment, ability to go to work, ability to have a mental circle of support.

And all of that for, you know, millions of Americans, like huge, huge impact. So that's one aspect of importance, so basically mental disorders. Alzheimer's has a huge importance in the well-being of Americans. Whether or not it dies, it kills someone. For many, many years, it has a huge impact. So the first measure of importance is just well-being. Like impact on the quality of life. Impact on the quality of life, absolutely. The second metric, which is much easier to quantify, is deaths.

What is the number one killer? The number one killer is actually heart disease. It is actually killing 650,000 Americans per year

Number two is cancer with 600,000 Americans. Number three, far, far down the list is accidents, every single accident combined. So basically, you know, you read the news accidents, like, you know, there was a huge car crash all over the news, but the number of deaths, number three by far, 167,000 lower respiratory disease. So that's asthma and not being able to breathe and so on and so forth. 160,000.

Alzheimer's, number five with 120,000, and then stroke, brain aneurysms, and so forth, that's 147,000, diabetes, and metabolic disorders, et cetera, that's 85,000. The flu is 60,000, suicide, 50,000, and then overdose, et cetera, you know, goes further down the list.

So of course, COVID has creeped up to be the number three killer this year with more than 100,000 Americans and counting. But if you think about what do we use, what are the most important diseases? You have to understand both the quality of life

and the sheer number of deaths, and just numbers of years lost, if you wish. And each of these diseases you can think of as, and also including terrorist attacks, as cool shootings, for example, things which lead to fatalities, you can look at as problems that could be solved.

and some problems are harder to solve than others. That's part of the equation. So maybe if you look at these diseases, if you look at heart disease or cancer or Alzheimer's or just schizophrenia and obesity, not necessarily things that kill you but affect the quality of life, which problems are solvable, which aren't, which are harder to solve, which aren't

I love your question because he puts it in the context of a global effort rather than just a local effort. So basically, if you look at the global aspect,

Exercise and nutrition are two interventions that we can as a society make a much better job at. So if you think about sort of the availability of cheap food, it's extremely high in calories, it's extremely detrimental for you, like a lot of processed food, et cetera. So if we change that equation and as a society, we made availability of healthy food much, much easier and charged a burger at McDonald's,

the price that it costs on the health system, then people would actually start buying more healthy foods. So basically, that's sort of a societal intervention, if you wish, in the same way.

increasing empathy, increasing education, increasing the social framework and support would basically lead to fewer suicides, it would lead to fewer murders, it would lead to fewer, you know, deaths overall. So, you know, that's something that we as a society can do. You can also think about external factors versus internal factors. So the external factors are basically communicable diseases like COVID, like the flu, et cetera. And

The internal factors are basically things like cancer and Alzheimer's, where your genetics will eventually drive you there.

And then, of course, with all of these factors, every single disease has both a genetic component and environmental component. So heart disease, huge genetic contribution. Alzheimer's, it's like 60% plus genetic. So I think it's like 79% heritability. So that basically means that genetics alone explains 79% of Alzheimer's incidence.

Yes, there's a 21% environmental component where you could basically enrich your cognitive environment, enrich your social interactions, read more books, learn a foreign language, go running, have a more fulfilling life. All of that will actually decrease Alzheimer's.

there's a limit to how much that can impact because of the huge genetic footprint. So this is fascinating. So each one of these problems have a genetic component and an environment component. And so when there's a genetic component, what can we do about some of these diseases? What have you worked on? What can you say that's in terms of problems that are solvable here or understandable?

So my group works on the genetic component, but I would argue that understanding the genetic component can have a huge impact even on the environmental component. Why is that? Because genetics gives us access to mechanism.

And if we can alter the mechanism, if we can impact the mechanism, we can perhaps counteract some of the environmental components. So understanding the biological mechanisms leading to disease is extremely important in being able to intervene. But when you can intervene, the analogy that I like to give is, for example, for obesity. Think of it as a giant bathtub of fat.

There's basically fat coming in from your diet, and there's fat coming out from your exercise. That's an in-out equation, and that's the equation that everybody's focusing on. But your metabolism impacts that bathtub. Basically, your metabolism controls the rate at which you're burning energy. It controls the rate at which you're storing energy.

And it also teaches you about the various valves that control the input and the output equation. So if we can learn from the genetics, the valves, we can then manipulate those valves

And even if the environment is feeding you a lot of fat and getting a little fat out, you can just poke another hole at the bathtub and just get a lot of the fat out. Yeah, that's fascinating. Yeah, so that we're not just passive observers of our genetics. The more we understand, the more we can come up with actual treatments.

And I think that's an important aspect to realize when people are thinking about strong effect versus weak effect variants. So some variants have strong effects. We talked about these Mendelian disorders where a single gene has a sufficiently large effect, pen and transics, percivity, and so on and so forth, that basically you can trace it in families with cases and not cases, cases, not cases, and so forth.

But these are the genes that everybody says, oh, that's the genes we should go after, because that's a strong effect gene. I like to think about it slightly differently. These are the genes where genetic impacts that have a strong effect were tolerated.

Because every single time we have a genetic association with disease, it depends on two things. Number one, the obvious one, whether the gene has an impact on the disease. Number two, the more subtle one, is whether there is genetic variation, standing and circulating and segregating in the human population that impacts that gene. Some genes are so darn important that if you mess with them even a tiny little amount,

That person is dead. So those genes don't have variation. You're not going to find a genetic association if you don't have variation. That doesn't mean that the gene has no role. It simply means that the gene tolerates no mutations. So that's actually a strong signal when there's no variation. That's so fast. Exactly. Genes that have very little variation are hugely important. You can actually rank the importance of genes based on how little variation they have. And those genes that have very little variation

but no association with disease, that's a very good metric to say, oh, that's probably a developmental gene because we're not good at measuring those phenotypes. So it's genes that you can tell evolution has excluded mutations from, but yet we can't see them associated with anything that we can measure nowadays. It's probably early embryonic lethal. What are all the words you just said early embryonic what lethal meaning

Meaning that you don't have that guy. Okay. There's a bunch of stuff that is required for a stable functional organism across the board for our entire species, I guess. If you look at sperm, it expresses thousands of proteins. Does sperm actually need thousands of proteins? No, but it's probably just testing them.

So my speculation is that misfolding of these proteins is an early test for failure. So that out of the millions of sperm that are possible, you select the subset that are just not grossly misfolding thousands of proteins.

So it's kind of an assert that this is fully correctly. Correct. Yeah, this just because if this little thing about the folding of a protein isn't correct, that probably means somewhere down the line, there's a bigger issue. That's exactly right. So fail fast. So basically, if you look at the mammalian investment in a new born, that investment is enormous in terms of resources.

So mammals have basically evolved mechanisms for fail fast. We're basically in those early months of development. I mean, it's horrendous, of course, at the personal level when you lose your future child, but in some ways,

There's so little hope for that child to develop and sort of make it through the remaining months that sort of fail fast is probably a good evolutionary principle for mammals. And of course, humans have a lot of medical resources that you can sort of give those children a chance.

And we have so much more success in giving folks who have these strong carrier mutations a chance. But if they're not even making it through the first three months, we're not going to see them. So that's why when we say what are the most important genes to focus on, the ones that have a strong effect mutation or the ones that have a weak effect mutation,

Well, you know, the jury might be out because the ones that have a strong effect mutation are basically, you know, not mattering as much. The ones that only have weak effect mutations

By understanding through genetics that they have a weak effect mutation and understanding that they have a causal role on the disease, we can then say, okay, great, evolution has only tolerated a 2% change in that gene. Pharmaceutical, I can go in and induce a 70% change in that gene. And maybe I will poke another hole at the bathtub that was not easy to control.

in, you know, many of the other sort of strong effect genetic variants. So this is this beautiful map of across the population of things that you're saying strong and weak effects are stuffed with a lot of mutations and stuff with little mutations with no mutations. And you have this map and it lays out the puzzle.

Yeah, so when I say strong effect, I mean at the level of individual mutations. So basically genes where, so you have to think of first the effect of the gene on the disease. Remember how I was sort of painting that map earlier from genetics all the way to phenotype? That gene can have a strong effect on the disease, but the genetic variant might have a weak effect on the gene.

So basically, when you ask what is the effect of that genetic variant on the disease, it could be that that genetic variant impacts the gene by a lot, and then the gene impacts the disease by a little, or it could be that the genetic variant impacts the gene by a little, and then the gene impacts the disease by a lot. So what we care about is genes that impact the disease a lot, but genetics gives us the full equation. And what I would argue is if we couple the genetics with

expression variation to basically ask what genes change by a lot and you know which genes correlate with disease by a lot even if the genetic variance change them by a little.

then that those are the best places to intervene. Those are the best places we're pharmaceutical. If I have even a modest effect, I will have a strong effect on the disease. Whereas those genetic variants that have a huge effect on the disease, I might not be able to change that gene by this much without affecting all kinds of other things. Interesting. So that's what we're looking at. Then what have we been able to find in terms of which disease could be helped?

Again, don't get me started. We have found so much. Our understanding of disease has changed so dramatically with genetics. I mean, places that we had no idea would be involved. So one of the worst things about my genome is that I have a genetic predisposition to age-related magular degeneration, AMD. So it's a form of blindness that causes you to lose the central part of your vision.

progressively as you grow older. My increased risk is fairly small. I have an 8% chance. You only have a 6% chance. I'm an average. Yeah. By the way, when you say my, you mean literally yours. You know this about you. I know this about me. Yeah. Which is kind of, uh,

I mean, philosophically speaking is a pretty powerful thing to live with. I mean, maybe that's what we agreed to talk again, by the way, for the listeners to where we're going to try to focus on science today and a little bit of philosophy next time. But it's interesting to think about the more you're able to know about yourself from the genetic information in terms of the diseases, how that changes your own view of life.

Yeah. So there's a lot of impact there. And there's something called genetics exceptionalism, which basically thinks of genetics as something very, very different than everything else as a type of determinism. And, you know, let's talk about that next time. So basically... That's a good preview. Yeah. So let's go back to AMD. So basically with AMD, we have no idea what causes AMD.

You know, it was a mystery until the genetics were worked out. And now the fact that I know that I have a predisposition allows me to sort of make some life choices, number one. But number two, the genes that lead to that predisposition give us insights as to how does it actually work. And that's a place where genetics gave us something totally unexpected. So there's a complement pathway

which is an immune function pathway that was in most of the loci associated with AMD. And that basically told us that, wow, there's an immune basis to this eye disorder that people had just not expected before. If you look at complement, it was recently also implicated in schizophrenia.

And there's a type of microglia that is involved in synaptic pruning. So synapses are the connections between neurons. And in this whole user or lucid view of mental cognition and other capabilities, you basically have microglia, which are immune cells, that are sort of constantly traversing your brain. And then pruning, neuronal connections, pruning synaptic connections, that are not utilized.

So in schizophrenia, there's thought to be a change in the pruning. That basically, if you don't prune your synapses the right way, you will actually have an increased role of schizophrenia. This is something that was completely unexpected for schizophrenia. Of course, we knew it has to do with neurons, but the role of the complement complex, which is also implicated in AMD, which is now also implicated in schizophrenia was a huge surprise. What's the complement complex?

So it's basically a set of genes, the complement genes, that are basically having various immune roles. And as I was saying earlier, our immune system has been co-opted for many different roles across the body. So they actually play many diverse roles. And somehow the immune system is connected to the synaptic pruning process. Exactly. So immune cells were co-opted to prune synapse. How did you figure this out?

How does one go off of this intricate connection pipeline of connections out? Yeah, let me give you another example. So Alzheimer's disease, the first place that you would expect it to act is obviously the brain. So we had basically this roadmap epigenomics consortium view of the human epigenome, the largest map of the human epigenome that has ever been built.

across 127 different tissues and samples with dozens of epigenomic marks measured in hundreds of donors. So what we've basically learned through that is that you basically can map what are the active gene regulatory elements for every one of the tissues in the body. And then we connected these gene regulatory active maps of basically what regions of the human genome are turning on in every one of different tissues.

We then can go back and say, where are all the genetic loci that are associated with disease? This is something that my group, I think, was the first to do back in 2010 in this Ernst Nature biotech paper. But basically, we were, for the first time, able to show that specific chromatin states, specific epigenomic states, in that case, enhancers, were, in fact, enriched in disease-associated variants.

We pushed that further in the Ernst nature paper a year later. And then in this roadmap, maybe genomics paper, you know, if you're, if you're after that, but basically that matrix that you mentioned earlier was in fact, the first time that we could see what genetic traits have genetic variants that are enriched in what tissues in the body.

And a lot of that map made complete sense. If you looked at a diversity of immune traits like allergies and type 1 diabetes and so forth, you basically could see that they were enriching

that the genetic variants associated with those traits were enriched in enhancers in these gene regulatory elements, active in T cells and B cells and hematopoietic stem cells and so on and so forth. So that basically gave us confirmation in many ways that those immune traits were indeed enriched in immune cells. If you looked at type 2 diabetes,

You basically saw an enrichment in only one type of sample, and it was pancreatic islets. And we know that type 2 diabetes sort of stems from the dysregulation of insulin in the beta cells of pancreatic islets. And that sort of was spot on super precise.

If you looked at blood pressure, where would you expect blood pressure to occur? You know, I don't know, maybe in your metabolism, in ways that you process coffee or something like that. Maybe in your brain, the way that you stress out and increases your blood pressure, et cetera. What we found is that blood pressure localized specifically in the left ventricle of the heart. So the enhancers of the left tectronol in the heart contain a lot of genetic variants associated with blood pressure. If you look at height,

we found an enrichment specifically in embryonic stem cell enhancers. So the genetic variants predisposing you to be taller or shorter are in fact acting in developmental stem cells makes complete sense.

If you looked at inflammatory bowel disease, you basically found inflammatory, which is immune, and also bowel disease, which is digestive. And indeed, we saw a double enrichment, both in the immune cells and in the digestive cells. So that basically told us that this is acting in both components. There's an immune component to inflammatory bowel disease, and there's a digestive component. And the big surprise was for Alzheimer's, we had seven different brain samples

We found zero enrichment in the brain samples for genetic variants associated with Alzheimer's. And this is mind-boggling. Our brains were literally hurting. What is going on? And what is going on is that the brain samples are primarily neurons, oligodendrocytes and astrocytes in terms of the cell types that make them up.

So that basically indicated that genetic variants associated with Alzheimer's were probably not acting in oligosinocytes, astrocytes or neurons. So what could they be acting in? Well, the fourth major cell type is actually microglia. Microglia are resident immune cells in your brain.

They immune a while. And they are CD14+, which is this sort of cell surface markers of those cells. So they're CD14+, cells, just like microfages that are circulating in your blood.

The microglia are resident monocytes that are basically sitting in your brain. They're tissue-specific monocytes. And every one of your tissues, like your fat, for example, has a lot of macrophages that are resident. And the M1 versus M2 macrophage ratio has a huge role to play in obesity. And so basically, again, these immune cells are everywhere. But basically what we found through this completely unbiased view of what are the tissues that likely underlie different disorders,

We found that Alzheimer's was humongously enriched in microglia, but not at all in the other cell types.

Tissue is involved. Is that simply useful for indication of propensity for disease? Or does it give us somehow a pathway of treatment? It's very much the second. If you look at the way to therapeutics, you have to start somewhere. What are you going to do? You're going to basically make assays that manipulate

those genes and those pathways in those cell types. So before we know the tissue of action, we don't even know where to start. We basically are at a loss, but if you know the tissue of action, and even better if you know the pathway of action, then you can basically screen your small molecules. Not for the gene. You can screen them directly for the pathway.

in that cell type. So you can basically develop a high throughput multiplexed robotic system for testing the impact of your favorite molecules that you know are safe, efficacious, and sort of hit that particular gene and so on and so forth. You can basically screen those molecules against either a set of genes that act in that pathway or on the pathway directly by having a cellular assay.

And then you can basically go into mice and do experiments and basically sort of figure out ways to manipulate these processes that allow you to then go back to humans and do a clinical trial that basically says, okay, I was able indeed to reverse these processes in mice. Can I do the same thing in humans?

So that the knowledge of the tissues gives you the pathway to treatment, but that's not the only part. There are many additional steps to figuring out the mechanism of disease. So that's really promising. Maybe take a small step back. You've mentioned all these puzzles that were figured out with the nature paper,

For me, you've mentioned a ton of diseases from obesity to Alzheimer's, even schizophrenia, I think you mentioned. What is the actual methodology of figuring this out? Indeed, I mentioned a lot of diseases. My lab works on a lot of different disorders. The reason for that is that if you look at the

If you look at biology, it used to be zoology departments and botanology departments and, you know, virology departments and so on and so forth. And MIT was one of the first schools to basically create a biology department like, oh, we're going to study all of life suddenly. Why was that even a case? Because the advent of DNA and the genome and the central dogma of DNA makes RNA makes protein in many ways unified biology.

you could suddenly study the process of transcription in viruses or in bacteria and have a huge impact on yeast and fly and maybe even mammals because of this realization of these common underlying processes. And in the same way that DNA unified biology, genetics is unifying disease studies. So you used to have

You used to have, I don't know, cardiovascular disease department and neurological disease department and neurodegeneration department and basically immune and cancer and so on and so forth. And all of these were studied in different labs.

because it made sense, because basically the first step was understanding how the tissue functions and we kind of knew the tissues involved in cardiovascular disease and so on and so forth. But what's happening with human genetics is that all of these walls and edifices that we had built are crumbling. And the reason for that is that genetics is in many ways revealing unexpected connections. So suddenly we now have to bring the immunologists to work on Alzheimer's.

They were never in their room. They were in another building altogether. The same way for schizophrenia, we now have to sort of worry about all these interconnected aspects. For metabolic disorders, we're finding contributions from brain. So suddenly we have to call the neurologist from the other building and so on and so forth. So in my view, it makes no sense anymore to basically say, oh, I'm a geneticist studying immune disorders.

I mean, that's ridiculous because, I mean, of course, in many ways, you still need to sort of focus. But what we're doing is that we're basically saying we'll go wherever the genetics takes us.

And by building these massive resources, by working on our latest maps, now 833 tissues, sort of the next generation of the epigenomics roadmap, which we're now called EpiMap, is 833 different tissues. And using those, we've basically found enrichments in 540 different disorders. Those enrichments are not like, oh, great, you guys work on that and we'll work on this.

They're intertwined amazingly. So, of course, there's a lot of modularity, but there's these enhancers that are sort of broadly active and these disorders that are broadly active. So, basically, some enhancers are active in on tissues and some disorders are enriching in on tissues. So, basically, there's these multifactorial and these other class, which I like to call polyfactorial diseases, which are basically lighting up everywhere.

And in many ways, it's cutting across these walls that were previously built across these departments. And the polyfactorial ones were probably the previous structure departments wasn't equipped to deal with those.

Again, maybe it's a romanticized question, but you know, there's in physics, there's a theory of everything. Do you think it's possible to move towards an almost theory of everything of disease from a genetic perspective? So if this unification continues, is it possible that, like, do you think in those terms, like trying to arrive at a fundamental understanding of how disease emerges, period? That unification is not just

foreseeable, it's inevitable. I see it as inevitable. We have to go there. You cannot be a specialist anymore if you're a genomicist. You have to be a specialist in every single disorder. And the reason for that is that the fundamental understanding of the circuitry of the human genome that you need to solve schizophrenia,

That fundamental circuitry is hugely important to solve Alzheimer's. And that same circuitry is hugely important to solve metabolic disorders. And that same exact circuitry is hugely important for solving immune disorders and cancer and, you know, every single disease. So all of them have the same sub-task. And I teach dynamic programming in my class

Dynamic program is all about sort of not Redoing the work. It's reusing the work that you do once so basically for us to say oh great You know you guys in the immune building go solve the fundamental circuitry of everything and then you guys in the schizophrenia building go solve the fundamental circuitry of everything separately It's crazy. So what we need to do is come together and sort of have a circuitry group the circuitry building that sort of tries to solve the circuitry of everything and

And then the immune folks who will apply this knowledge to all of the disorders that are associated with immune dysfunction. And the schizophrenia folks will basically interact with both the immune folks and with the neuronal folks. And all of them will be interacting with the circuitry folks and so on and so forth. So that's sort of the current structure of my group, if you wish. So basically what we're doing is focusing on the fundamental circuitry.

But at the same time, we're the users of our own tools by collaborating with many other labs in every one of these disorders that we mentioned. We basically have a heart focus on cardiovascular disease, coronary artery disease, heart failure, and so on and so forth.

We have an immune focus on several immune disorders. We have a cancer focus on metastatic melanoma and immunotherapy response. We have a psychiatric disease focus on schizophrenia, autism, PTSD, and other psychiatric disorders. We have an Alzheimer's and neurodegeneration focus.

on hunting to the disease, ALS, and AD related disorders like frontotemporal dementia and lewy body dementia, and of course a huge focus on Alzheimer's. We have a metabolic focus on the role of exercise and diet and sort of how they're impacting metabolic organs across the body and across many different issues. And all of them are interfacing with the circuitry

And the reason for that is another computer science principle of eat your own dog food. If everybody ate their own dog food, dog food would taste a lot better. The reason why Microsoft Excel and PowerPoint was so important and so successful is because the employees that were working on them were using them for their day-to-day tasks.

You can't just simply build a circuitry and say, here it is guys, take the circuitry we're done without being the hugers of that circuitry because you then go back and because we span the whole spectrum from profiling the epigenome, using comparative genomics, finding the important nucleotide in the genome,

Building the basic functional map of what are the genes in the human genome? What are the gene regulatory elements of the human genome? I mean over the years we've written a series of papers on how do you find human genes in the first place? Using comparative geomics. How do you find the motifs that are the building blocks of gene regulation? Using comparative genomics. How do you then find how these motifs come together?

and act in specific tissues using epigenomics. How do you link regulators to enhancers and enhancers to their target genes using epigenomics and regulatory genomics? So through the years we've basically built all this infrastructure for understanding what I like to say every single nucleotide of the human genome.

and how it acts in every one of the major cell types and tissues of the human body. I mean, this is no small task. This is an enormous task that takes the entire field, and that's something that my group has taken on, along with many other groups.

And we have also, and that sort of thing sets my group perhaps apart, we have also worked with specialists in every one of these disorders to basically further our understanding all the way down to disease. And in some cases, collaborating with pharma to go all the way down to therapeutics because of our deep, deep understanding of that basic circuitry and how it allows us to now improve the circuitry.

not just treat it as a black box, but basically go and say, okay, we need a better cell type specific wiring that we now have at the tissue specific level. So we're focusing on that because we're understanding the needs from the disease front.

Maybe you can indulge me in one nice question to ask would be, how do you, from the scientific perspective, go from knowing nothing about the disease to going, you said, to going through the entire pipeline and actually have a drug or a treatment that cures that disease.

So that's an enormously long path and an enormously great challenge. And what I'm trying to argue is that it progresses in stages of understanding rather than one gene at a time. The traditional view of biology was you have one postdoc working on this gene and another postdoc working on that gene. And they'll just figure out everything about that gene. And that's their job.

what we've realized is how polygenic the diseases are. So we can't have one post-doc protein anymore. We now have to have these cross-cutting needs. And I'm going to describe the path to circuitry along those needs. And every single one of these paths, we are now doing in parallel across thousands of genes. So the first step is you have a genetic association

And we talked a little bit about sort of the Mendelian path and the polygenic path to that association. So the Mendelian path was looking through families to basically find gene regions and ultimately genes that are underlying particular disorders. The polygenic path

It's basically looking at unrelated individuals in this giant matrix of genotype by phenotype, and then finding hits where a particular variant impacts disease all the way to the end. And then we now have a connection not between a gene and a disease, but between a genetic region and a disease.

And that distinction is not understood by most people, so I'm going to explain it a little bit more. Why do we not have a connection between a gene and a disease, but we have a connection between a genetic region and a disease? The reason for that is that 93% of genetic variants that are associated with disease don't impact the protein at all.

So if you look at the human genome, there's 20,000 genes, there's 3.2 billion nucleotides. Only 1.5% of the genome codes for proteins. The other 98.5% does not code for proteins. If you now look at where are the disease variants located?

93% of them fall in that outside the gene's portion. Of course, genes are enriched, but they're only enriched by a factor of 3. That means that still 93% of genetic variants fall outside the proteins. Why is that difficult? Why is that a problem? The problem is that when a variant falls outside the gene,

You don't know what gene is impacted by that variant. You can't just say, oh, it's near this gene. Let's just connect that variant to the gene. And the reason for that is that the genome circuitry is very often long range.

So you basically have that genetic variant that could sit in the intron of one gene. And an intron is sort of the place between the exons that code for proteins. So proteins are split up into exons and introns. And every exon codes for a particular subset of amino acids and together they're spliced together and then make the final protein.

So that genetic variant might be sitting in an intern of a gene. It's transcribed with a gene, it's processed and then excised, but it might not impact this gene at all. It might actually impact another gene that's a million nucleotides away. So it's just riding along even though it has nothing to do with a nearby neighbourhood. That's exactly right. Let me give you an example. The strongest genetic association with obesity was discovered in this FTO gene, fat and obesity associated gene.

So this FTO gene was studied ad nauseam. People did tons of experiments on it. They figured out that FTO is in fact RNA methylation transferase. It basically sort of impacts something that we know that we call the EPI transcriptome, just like the genome can be modified, the transcriptome, the transcripts of the genes.

can be modified. And we basically said, oh, great. That means that epi transcriptomics is hugely involved in obesity because that gene FTO is clearly where the genetic locus is at.

My group studied FTO in collaboration with, you know, a wonderful team led by Melina Klassner. And what we found is that this FTO locus, even though it is as associated with obesity, does not implicate the FTO gene.

The genetic variant sits in the first intran of the FTO gene, but it controls two genes, IRX3 and IRX5, that are sitting 1.2 million nucleotides away, several genes away.

Oh boy. Ah, what am I supposed to feel about that? Because isn't that like super complicated then? So the way that I was introduced at a conference a few years ago was, and here's my knowledge, Kelly's, who wrote the most depressing paper of 2015.

And the reason for that is that the entire pharmaceutical industry was so comfortable that there was a single gene in that locus. Because in some loci, you basically have three dozen genes that are all stating the same region of association. And you're like, gosh, which one of those is it? But even that question of which one of those is it is making the assumption that it is one of those, as opposed to some random gene just far, far away, which is what our paper showed.

So basically what our paper showed is that you can't ignore the circuitry. You have to first figure out the circuitry, all of those long range interactions, how every genetic variant impacts the expression of every gene in every tissue imaginable across hundreds of individuals.

And then you now have one of the building blocks, not even all of the building blocks, for then going and understanding disease. So embrace the wholeness of the circuitry. Correct. So back to the question of starting knowing nothing to the disease and going to the treatment.

So what are the next steps? So you basically have to first figure out the tissue and then describe how you figure out the tissue. You figure out the tissue by taking all of these non-coding variants that are sitting outside proteins and then figuring out what are the epigenomic enrichments. And the reason for that, you know, thankfully, is that there is convergence that the same processes are impacted in different ways by different loci.

And that's a saving grace for our field. The fact that if I look at hundreds of genetic variants associated with Alzheimer's, they localize in a small number of processes.

Can you clarify why that's hopeful? So they show up in the same exact way in the specific set of processes. Yeah. So basically, there's a small number of biological processes that underlie, or at least that play the biggest role in every disorder. So in Alzheimer's, you basically have maybe 10 different types of processes. One of them is lipid metabolism. One of them is immune cell function. One of them is neuronal energetics.

So, these are just a small number of processes, but you have multiple lesions, multiple genetic perturbations that are associated with those processes. So, if you look at schizophrenia, it's excitatory neuron function. It's inhibitory neuron function. It's synaptic pruning. It's calcium signaling and so and so forth. So, when you look at disease genetics, you have one hit here and one hit there and one hit there and one hit there, completely different parts of the genome.

But it turns out all of those hits are calcium signaling proteins. Oh, cool. You're like, aha. That means that calcium signaling is important. So those people who are focusing on one doctor's at a time cannot possibly see that picture. You have to become a genomesis. You have to look at the omics, the om, the holistic picture to understand these enrichments.

But you mentioned the convergence thing. Whatever the thing associated with the disease shows up. So let me explain convergence. Convergence is such a beautiful concept. So you basically have these four genes that are converging on calcium signaling.

So that basically means that they are acting each in their own way, but together in the same process. But now, in every one of these low side, you have many enhancers controlling each of those genes.

That's another type of convergence where dysregulation of seven different enhancers might all converge on dysregulation of that one gene, which then converges on calcium signaling. And in each one of those enhancers, you might have multiple genetic variants distributed across many different people.

Everyone has their own different mutation, but all of these mutations are impacting that enhancer, and all of these enhancers are impacting that gene, and all of these genes are impacting this pathway, and all of these pathways are acting in the same tissue, and all of these tissues are converging together on the same biological process of schizophrenia.

And you're saying the saving grace is that that conversion seems to happen for a lot of these diseases. For all of them, basically that for every single disease that we've looked at, we have found an epigenomic enrichment. How do you do that? You basically have

all of the genetic variants associated with the disorder, and then you're asking for all of the enhancers active in a particular tissue. For 540 disorders, we've basically found that indeed there is an enrichment. That basically means that there is commonality, and from the commonality we can just get insights. So to explain in the mathematical terms,

We're basically building an empirical prior. We're using a Bayesian approach to basically say, great, all of these variants are equally likely in a particular locus to be important energy. So in a genetic locus, you basically have a dozen variants that are co-inherited because the way that inheritance works in the human genome is through all of these recombination events during meiosis.

You basically have, you know, you inherit maybe three chromosome three, for example, in your body. It's inherited from four different parts. One part comes from your dad, another part comes from your mom, another part comes from your dad, another part comes from your mom.

So basically the way that it, sorry, from your mom's mom. So you basically have one copy that comes from your dad and one copy that comes from your mom. But that copy that you got from your mom is a mixture of her maternal and her paternal chromosome. And the copy that you got from your dad is a mixture of his maternal and his paternal chromosome. So these break points that happen when chromosomes are lining up, lining up,

are basically ensuring, through these crossover events, they're ensuring that every child's cell during the process of meiosis, where you basically have one spermatozoid that basically couples with one ovule to basically create one egg to basically create the zygote.

You basically have half of your genome that comes from that and half of your genome that comes from mom. But in order to line them up, you basically have this cross-rove events. These cross-rove events are basically leading to co-inheritance of that entire block coming from your maternal grandmother and that entire block coming from your maternal grandfather. Over many generations, these cross-rove events don't happen randomly.

There's a protein called PRDM9 that basically guides the double-stranded breaks and then leads to these crossovers. And that protein has a particular preference to only a small number of hotspots of recombination, which then leads to a small number of breaks between these co-inheritance patterns. So even though there are 6 million variants, there are 6 million loci, this variation is inherited in blocks.

And every one of these blocks has like 2,000 genetic variants that are all associated. So in the case of FTO, it wasn't just one variant, it was 89 common variants that were all humongously associated with obesity. Which ones of those is the important one? Well, if you look at only one locus, you have no idea. But if you look at many loci, you basically say, aha,

All of them are enriching in the same epigenomic map. In that particular case, it was mesenchymal stem cells. So these are the progenitor cells that give rise to your brown fat and your white fat. Progenitor is like the early on developmental stem. So you start from one zygote, and that's a totipotent cell type. It can do anything. You then, you know, that cell divides, divides, divides.

And then every cell division is leading to specialization.

where you now have a mesodermal lineage, an ectodermal lineage, an endodermal lineage that basically leads to different parts of your body. The ectoderm will basically give rise to your skin, ecto means outside, derm is skin. So ectoderm, but it also gives rise to your neurons and your whole brain. So that's a lot of ectoderm. Mesoderm gives rise to your internal organs, including the vasculature and your muscle and stuff like that.

So, you basically have this progressive differentiation, and then if you look further down that lineage, you basically have one lineage that will give rise to both your muscle and your bone, but also your fat. And if you go further down the lineage of your fat, you basically have your white fat cells,

These are the cells that store energy. So when you eat a lot, but you don't exercise too much, there's an excess set of calories, excess energy. What do you do with those? You basically create, you spend a lot of that energy to create these high energy molecules, lipids.

which you can then burn when you need them on a rainy day. So that leads to obesity if you don't exercise and if you overeat. Because if your body is like, oh, great, I have all these calories, I'm going to store them. Oh, more calories. I'm going to store them too. Oh, more calories. And the 42% of European chromosomes

have a predisposition to storing fat, which was selected probably in the food scarcity periods. Like basically, as we were exiting Africa before and during the ice ages, there was probably a selection to those individuals who made it north to basically be able to store energy, a lot more energy.

So you basically now have this lineage that is deciding whether you want to store energy in your white fat or burn energy in your base fat. Turns out that your fat is, you know, we have such a bad view of fat. Fat is your best friend. Fat can both store all these excess lipids that would be otherwise circulating through your body and causing damage.

But it can also burn calories directly. If you have too much energy, you can just choose to just burn some of that as heat.

So basically when you're cold, you're burning energy to basically warm your body up and you're burning all these lipids and you're burning all these caters. So what we basically found is that across the board, genetic variants associated with obesity across many of these regions were all enriched repeatedly in mesenchymal stem cell enhancers. So that gave us a hint as to which of these genetic variants was likely driving this whole association.

And we ended up with this one-genetic variant called RS-142-1085.

And that genetic variant out of the 89 was the one that we predicted to be causal for the disease. So going back to those steps. First step is figure out the relevant tissue based on the global enrichment. Second step is figure out the causal variant among many variants in this linkage disequilibrium, in this co-inherited block between these recombination hotspots.

These boundaries of these inherited blocks. That's the second step. The third step is once you know that causal variant, try to figure out what is the motif that is disrupted by that causal variant. Basically, how does it act? Variants don't just disrupt elements, they disrupt the binding of specific regulators.

So basically the third step there was how do you find the motif that is responsible, like the gene regulatory word, the building block of gene regulation, that is responsible for that dysregulatory event. And the fourth step is finding out what regulator normally binds that motif and is now no longer able to bind.

And then once you have the regulator, can you then try to figure out how to what after developed how to fix it? That's exactly right. You now know how to intervene. You have basically a regulator. You have a gene that you can then perturb, and you say, well, maybe that regulator has a global role in obesity. I can perturb the regulator. Just to clarify, when we say perturb, on the scale of a human life, can a human being be helped

Of course. Yeah, I guess understanding is the first step. No, no, but perturbed basically means you now develop therapeutics pharmaceutical therapeutics against that, or you develop other types of intervention that affect the expression of that gene. What do pharmaceutical therapeutics look like when your understanding is on a genetic level? Yeah. Sorry if it's a dumb question. No, no, no, it's a brilliant question, but I want to save it for a little bit later when we start talking about therapeutics. Perfect.

We've talked about the first four steps. There's two more. So basically the first step is figure out, I mean, the zero step, the starting point is the genetics. The first step after that is figure out the tissue of action. The second step is figuring out the nucleotide that is responsible or set of nucleotides. The third step is figure out the motif and the upstream regulator number four. Number five and six is what are the targets?

So number five is great. Now I know the regulator, I know the motif, I know the tissue, and I know the variant. What does it actually do? So you have to now trace it to the biological process and the genes that mediate that biological process.

So knowing all of this can now allow you to find the target genes. How? By basically doing perturbation experiments, or by looking at the folding of the epigenome, or by looking at the genetic impact of that genetic variant on the expression of genes. And we use all three. So let me go through them. Basically, one of them is physical links. This is the folding of the genome onto itself.

How do you even figure out the folding? It's a little bit of a tangent, but it's a super awesome technology. Think of the genome as, again, this massive packaging that we talked about of taking two meters' worth of DNA and putting it in something that's

a million times smaller than two meters worth of DNA, that's a single cell. You basically have this massive packaging, and this packaging basically leads to the chromosome being wrapped around in sort of tight, tight ways. In ways, however, that are functionally capable of being reopened and re-closed. So I can then go in and figure out that folding by sort of chopping up the spaghetti soup

putting glue and ligating the segments that were chopped up but nearby each other, and then sequencing through these ligation events to figure out that this region of the chromosome, that region of the chromosome were near each other, that means they were interacting, even though they were far away on the genome itself. So that chopping up sequencing and re-gluing is basically giving you folds

of the genome that we call it. So can you backtrack? How does cutting it help you figure out which ones were close in the original folding? So you have a bowl of noodles. Go on. And in that bowl of noodles, some noodles are near each other. So throwing a bunch of glue, you basically freeze the noodles in place, throw in a cutter that chops up the noodles into little pieces.

Now, throw in some ligation enzyme that lets those pieces that were free, re-ligate near each other. In some cases, they re-ligate what you had just got, but that's very rare. Most of the time, they will re-ligate in whatever was proximal. You now have glued the red noodle that was crossing the blue noodle to each other.

you then reverse the glue, the glue goes away, and you just sequence the heck out of it. Most of the time, you'll find a red segment with, you know, a red segment, but you can specifically select for legation events that have happened that were not from the same segment by sort of marking them in a particular way, and then selecting those, and then you sequencing, you look for red with blue matches of sort of things that were glued that were not immediate, proximal to each other.

And that reveals the linking of the blue noodle and the red noodle. You're with me so far? Yeah. Good. So we've done these experiments. That's physical. That's a physical. That's step one of the physical. And what the physical revealed is topologically associated domains, basically big blocks of the genome that are topologically connected together. That's the physical. The second one is the genetic links.

It basically says, across individuals that have different genetic variants, how are their genes expressed differently? Remember before I was saying that the path between genetics and diseases is enormous, but we can break it up to look at the path between genetics and gene expression. So instead of using Alzheimer's as the phenotype, I can now use expression of IRX3 as the phenotype, expression of gene A.

And I can look at all of the humans who contain a G at that location and all the humans will contain a T at that location. And basically say, wow, turns out that the expression of the gene is higher for the T humans than for the G humans at that location. So that basically gives me a genetic link between a genetic variant, a locus, a region, and the expression of nearby genes.

Good on the genetic link. I think so. Awesome. So the third link is the activity link. What's an activity link? It basically says if I look across 833 different epigenomes, whenever this enhancer is active, this gene is active. That gives me an activity link between this region of the DNA and that gene.

And then the fourth one is perturbations where I can go in and blow up that region and see what are the genes that change in expression. Or I can go in and over activate that region and see what genes change in expression. So I guess that's similar to activity.

Yeah, so it's similar to activity. I agree, but it's causal rather than correlational. Again, I'm a little weird. No, no, you're 100% on. It's exactly the same. But the perturbation. Where I go and intervene, I basically take a bunch of cells. So you know CRISPR, right? CRISPR is this genome guidance and cutting mechanism. It's what George George likes to call genome vandalism.

So you basically are able to... You can basically take a guide RNA that you put into the CRISPR system, and the CRISPR system will basically use this guide RNA, scan the genome, find wherever there's a match, and then cut the genome. So, you know, I digress, but it's a bacterial immune defense system. So basically bacteria are constantly attacked by viruses.

But sometimes they win against the viruses and they chop up these viruses and remember as a trophy inside their genome, they have this low side, this CRISPR low side that basically stands for clustered repeats, interspersed, et cetera. So basically it's an interspersed repeats structure where basically you have a set of repetitive regions and then interspersed were these variable segments that were basically matching viruses. So when this was first discovered,

It was basically hypothesized that this is probably a bacteria immune system that remembers the trophies of the viruses that manage the kill. And then the bacteria pass on, you know, they sort of do lateral transfer of DNA, and they pass on these memories so that the next bacterium says, oh, you kill that guy. When that guy shows up again, I will recognize him.

And the CRISPR system was basically evolved as a bacterial adaptive immune response to sense foreigners that should not belong and to just go and cut their genome. So it's an RNA-guided RNA-cutting enzyme or an RNA-guided DNA-cutting enzyme. So there's different systems, some of them cut DNA, some of them cut RNA, but all of them remember this sort of viral attack.

So what we have done now as a field is, you know, through the work of, you know, Jennifer Dunna, Manuel Carpontier, Feng Zhang, and many others, is co-opted that system of bacterial immune defense as a way to cut genomes. You basically have this guiding system that allows you to use an RNA guide to bring enzymes to cut DNA at a particular location.

That's so fascinating. So this is like already a natural mechanism, a natural tool for cutting those useful in this particular context. And we're like, well, we can use that thing to actually, it's a nice tool that's already in the body. Yeah. Yeah. It's not in our body. It's in the bacterial body. It was discovered by the, by the yogurt industry. They were trying to make better yogurts and they were trying to make their bacteria in their yogurt cultures more resilient to viruses.

And they were studying bacteria, and they found that, wow, this CRISPR system is awesome. It allows you to defend against that. And then it was co-opted in mammalian systems that don't use anything like that as a targeting way to basically bring these DNA cutting enzymes to any location in the genome. Why would you want to cut DNA?

to do anything. The reason is that our DNA has a DNA repair mechanism, where if a region of the genome gets randomly cut, you will basically scan the genome for anything that matches

and sort of use it by homology. So the reason why we're deployed is because we now have a spare copy. As soon as my mom's copy is deactivated, I can use my dad's copy. And somewhere else, if my dad's copy is deactivated, I can use my mom's copy to repair it. So this is called homologous based repair.

So all you have to do is the cutting and you don't have to do the fixing. That's exactly right. You don't have to do the fixing. Because it's already built in. That's exactly right. But the fixing can be co-opted by throwing in a bunch of homologous segments that instead of having your dad's version, have whatever other version you'd like to use.

So you then control the fixing by throwing in a bunch of other stuff. That's exactly right. And that's how you do genome editing. So that's what CRISPR is. That's what CRISPR is. One in popular culture people use the term. I've never, wow, that's brilliant. So CRISPR is genome vandalism followed by a bunch of Band-Aids that have the sequence that you'd like. And you can control the choices of Band-Aids. Correct. Yeah.

And of course, there's new generations of CRISPR. There's something that's called prime editing that was sort of very much in the press recently, that basically instead of sort of making a double-stranded break, which again is genome vandalism, you basically make a single stranded break, you basically just nick one of the two strands, enabling you to sort of peel off without sort of completely breaking it up.

and then repair it locally using a guide that is coupled to your initial RNA that took you to that location. Dumb question, but is CRISPR as awesome and cool as it sounds? I mean, technically speaking, in terms of like, as a tool for manipulating our genetics in the positive meaning of the word manipulating.

Or is there downsides, drawbacks in this whole context of therapeutics that we're talking about or understanding and so forth? So when I teach my students about CRISPR, I show them articles with the headline, genome editing tool revolutionizes biology, and then I show them the date of these two of these articles, and they're 2004.

like five years before CRISPR was invented. And the reason is that they're not talking about CRISPR. They're talking about zinc finger enzymes. There are another way to bring these coders to the genome. It's a very difficult way of sort of designing the right set of zinc finger proteins, the right set of amino acids that will now target a particular long stretch of DNA.

Because for every location that you want to target, you need to design a particular regulator, a particular protein, that will match that region. Well, there's another technology called tailons, which are basically just a different way of using proteins to guide these coders to a particular location of the genome.

These require a massive team of engineers, of biological engineers, to basically design a set of amino acids that will target a particular sequence of your genome. The reason why CRISPR is amazingly awesomely revolutionary is because instead of having this team of engineers design a new set of proteins for every locker that you want to target, you just type it in your computer and you just synthesize an RNA guide.

The beauty of CRISPR is not the cutting. It's not the fixing. All of that was there before. It's the guiding. And the only thing that changes is that it makes the guiding easier by sort of, you know, just typing in the RNA sequence, which then allows the system to sort of scan the DNA to find that.

So the coding, the engineering of the cutter is easier in terms of SB. That's kind of similar to the story of deep learning versus old school machine learning. Some of the challenging parts are automated. Okay, so, but Chris, there's just one cutting technology. And then there's, well, that's part of the challenges and exciting opportunities of the field. It's the design different cutting technologies.

Now, this was a big parentheses on CRISPR, but now, when we were talking about perturbations, you basically now have the ability to not just look at correlation between enhancers and genes, but actually go and either destroy that enhancer and see if the gene changes in expression, or you can use the CRISPR targeting system

to bring in not vandalism and cutting, but you can couple the CRISPR system with, and the CRISPR system is called usually CRISPR-Cas9, because Cas9 is the protein that will then come and cut. But there's a version of that protein called DeadCas9, where the cutting part is deactivated. So you basically use DCas9, DeadCas9, to bring in an activator, or to bring in a repressor.

So you can now ask, is this enhancer changing that gene by taking this modified CRISPR, which is already modified from the bacteria to be used in humans, that you can now modify the Cas9 to be dead Cas9, and you can now further modify it to bring in a regulator. And you can basically turn on or turn off that enhancer and then see what is the impact on that gene. So these are the four ways of linking the locus to the target gene. And that's step number five.

Okay, step number five is find the target gene. And step number six is what the heck does that gene do? You basically now go and manipulate that gene to basically see what are the processes that change.

And you can basically ask, well, you know, in this particular case, in the FDA locus, we found mesenchymal stem cells that are the progenitors of white fat and brown fat or beige fat. We found the RS-1421085 nucleotide variant as the causal variant. We found this large enhancer, this master regulator. I like to call it OB-1, for obesity-1, like the strongest enhancer associated with it.

and Obi-Wan was kind of chubby as the actor and if you remember him.

Yeah. So you basically are using this Jedi mind trick to basically find out the... Thank you. The location of the genome that is responsible, the enhancer that harbors it, the motif, the upstream regulator, which is arid 5B for AT-rich interacting domain 5B. That's a protein that sort of comes and binds normally. That protein is normally a repressor. It represses this super enhancer, this massive 12,000 nucleotide master regulatory control region.

And it turns off IRX3, which is a gene that's 600,000 nucleotides away, and IRX5, which is 1.2 million nucleotides away. So those... And what's the effect of turning them off? That's exactly the next question. So step six is, what do these genes actually do? So we then ask, what does IRX3 and IRX5 do? The first thing we did is look across individuals for individuals that had higher expression of IRX3 or lower expression IRX3. And then we looked at the expression of all of the other genes in the genome.

And we looked for simply correlation. And we found that iRx3 and iRx5 were both correlated positively with lipid metabolism and negatively with mitochondrial biogenesis. You're like, what the heck does that mean?

Doesn't sound related to obesity? Not at all, superficially. But lipid metabolism should, because lipids is these high-energy molecules that basically store fat. So, higher exteninitis V are negatively correlated with lipid metabolism. So, that basically means that when they turn on, when they turn on, they turn on lipid metabolism. And they're negatively correlated with

mitochondrial biogenes. What do mitochondria do in this whole process? Again, small parenthesis. What are mitochondria? Mitochondria are little organelles.

They arose. They only are found in eukaryotes. U means good, carrier means nucleus, so truly like a true nucleus. So eukaryotes have a nucleus. Prokaryotes are before the nucleus. They don't have a nucleus. So eukaryotes have a nucleus. Hm, compartmentalization. Eukaryotes have also organelles. Some eukaryotes have chloroplasts. These are the plants. They photosynthesize.

Some other eukaryotes, like us, have another type of organelle called mitochondria. These arose from an ancient species that we engulfed.

This is an endosymbiosis event. Symbiosis, bio means life, sim means together. So symbiotes are things that live together. Endosymbiosis, endo means inside. So endosymbiosis means you live together, holding the other one inside you. So the pre-ukaryotes engulfed an organism that was very good at energy production.

And that organism eventually shed most of its genome to now have only 13 genes in the mitochondrial genome. And those 13 genes are all involved in energy production, the electron transport chain. So basically, electrons are these massive super energy rich molecules.

We basically have these organelles that produce energy. And when your muscle exercises, you basically multiply your mitochondria. You basically sort of use more and more mitochondria. And that's how you get beefed up. You basically, the muscle sort of learns how to generate more energy. So basically every single time your muscles will overnight, regenerate, and sort of become stronger and amplify their mitochondrions and so forth.

So what do the mitochondria do? The mitochondria use energy to sort of do any kind of task. When you're thinking, you're using energy. This energy comes from mitochondria. Your neurons have mitochondria all over the place. Basically, this mitochondria can multiply its organelles and they can be spread along the body of your muscle.

Some of your muscle cells have actually multiple nuclei. They're probably nucleated, but they also have multiple mitochondria to basically deal with the fact that your muscle is enormous. You can sort of span these super, super long length and you need energy throughout the length of your muscle. So that's why you have mitochondria throughout the length. And you also need transcription through the length. So you have multiple nuclei as well. So these two processes, lipids, store energy, what do mitochondria do? So there's a process known as thermal

Genesis. Thermo heat, Genesis generation. Thermo Genesis is a generation of heat. Remember that bathtub?

with the in and out, that's the equation that everybody's focused on. So how much energy do you consume? How much energy do you burn? But in every thermodynamic system, there's three parts to the equation. There's energy in, energy out, and energy lost. Any machine has loss of energy. How do you lose energy? You emanate heat. So heat is energy loss.

There's... Which is where the thermogenesis comes in. Thermogenesis is actually a regulatory process that modulates the third component of the thermodynamic equation. You can basically control thermogenesis explicitly. You can turn on and turn off thermogenesis. And that's where the mitochondria comes into. Exactly. So, IRX, DNRX5, turn out to be the master regulators of a process of thermogenesis versus lipogenesis, generational fat.

So, RX10 and RX5 in most people burn heat, burn calories as heat. So, when you eat too much, just burn it off in your fat cells. So, with that bathtub, that's basically a dissipation knob that most people are able to turn on. I am unable to turn that on.

because I am a homozygous carrier for the mutation that changes the T into a C in the RS-1-4-2-1-0-8-5 allele, a locus, a SNP. I have the risk allele twice from my mom and from my dad. So I'm unable to thermogenize.

I'm able to turn on thermogenesis through IRX-3 and IRX-5 because the regulator that normally binds here, IRX-5V, can no longer bind because it's an AT-rich interacting domain. And as soon as I change the T into a C, it can no longer bind because it's no longer AT-rich.

But doesn't that mean that you're able to use energy more efficiently? You're not generating heat? Or is that true? That means I can eat less and get around just fine. Yes. Yeah. So that's a feature, actually. It's a feature in a food scars environment. Yeah. But if we're all starving, I'm doing great.

If we all have access to massive amounts of food, I'm obese, basically. That's taken us through the entire process of then understanding that why mitochondria and then lipids are both even though distant are somehow involved. Different sides of the same coin. You basically choose to store energy or you can choose to burn energy. All of that is involved in the puzzle of obesity.

And that was fascinating, right? Here we are in 2007, discovering the strongest genetic association with obesity, and knowing nothing about how it works for almost 10 years. For 10 years, everybody focused on this FTO gene.

And they were like, oh, it must have to do something with, you know, RNA modification. And it's like, no, it has nothing to do with the function of FDO. It has everything to do with all of these other processes. And suddenly, the moment you solve that puzzle, which is a multi-year effort, by the way, and a tremendous effort by Melina and many, many others.

So this tremendous effort basically led us to recognize this circuitry. You went from having some 89 common variants associated in that region of the DNA sitting on top of this gene to knowing the whole circuitry.

When you know the circuitry, you can now go crazy. You can now start intervening at every level. You can start intervening at the iris 5b level. You can start intervening with CRISPR-Cas9 at the single SNP level. You can start intervening at iris 3 and iris 5 directly there. You can start intervening at the thermogenesis level because you know the pathway. You can start intervening at the differentiation level where these

the decision to make either white fat or beige fat, the energy burning beige fat, is made developmentally in the first three days of differentiation of your adipocytes. So as they're differentiating, you basically can choose to make fat burning machines or fat storing machines. And sort of that's how you populate your fat. You basically can now go in from a suitically and do all of that. And in our paper, we actually did all of that. We went in and manipulated every single aspect.

At the nucleotide level, we use CRISPR-Cas9 genome editing to basically take primary adipocytes from risk and non-risk individuals and show that by editing that one nucleotide out of 3.2 billion nucleotides in the human genome, you could then flip between an obese phenotype and a lean phenotype like a switch. You can basically take my cells that are non-thermogenizing and just flip into thermogenizing cells, but change your one nucleotide. It's mind-boggling.

It's so inspiring that this puzzle could be solved in this way, and it feels within reach to then be able to crack the problem of some of these diseases. So 2007, you mentioned 2000. What are the technologies, the tools that came along that made this possible? What are you excited about? Maybe if we just look at the buffet of things that you've kind of mentioned.

What's involved? What should we be excited about? What are you excited about? I love that question because there's so much ahead of us. There's so, so basically solving that one locus required massive amounts of knowledge that we have been building across the years through the epigenome, through the comparative genomics to find out the causal variant and the controller regulator motif through the conserved circuitry.

It required knowing this regulatory genomic wiring. It required high C of the sort of topologically associated domains to basically find this long range interaction. It required ECTLs of the sort of genetic perturbation of these intermediate gene phenotypes. It required all of the arsenal of tools that have been describing was put together for one locus.

And this was a massive team effort, huge investment in time, energy, money, effort, intellectual, everything. You're referring to, I'm sorry. This one paper. Yeah, this one single paper. This one single paper. I like to say that this is a paper about one nucleotide in the human genome, about one bit of information, C versus T in the human genome. That's one bit of information, and we have 3.2 billion nucleotides to go through.

So how do you do that systematically? I am so excited about the next phase of research because the technologies that my group and many other groups have developed allows us to now do this systematically, not just one locus at a time, but thousands of loci at a time. So let me describe some of these technologies.

The first one is automation and robotics. So basically, you know, we talked about how you can take all of these molecules and see which of these molecules are targeting each of these genes and what do they do? So you can basically now screen through millions of molecules, through thousands and thousands and thousands of plates, each of which has thousands and thousands and thousands of molecules. Every single time testing, you know, all of these genes,

and asking which of these molecules perturbed these genes. So that's technology number one, automation and robotics. Technology number two is parallel readouts. So instead of perturbing one locus,

and then asking if I use CRISPR-Cas9 on this enhancer to basically use D-Cas9 to turn on or turn off the enhancer, or if I use CRISPR-Cas9 on the SNP to basically change that one SNP at a time, then what happened? But we have 120,000 disease-associated SNPs that we want to test. We don't want to spend 120,000 years doing it. So what do we do?

And we've basically developed this technology for massively parallel reporter assays, M-P-R-A. So in collaboration with Tarjan Mickelson, Eric Lander, I mean, Jason Duhrs' group has done a lot of that. So there's a lot of groups that basically have developed technologies for testing 10,000 genetic variants at a time.

How do you do that? We talked about microarray technology, the ability to synthesize these huge microarrays that allow you to do all kinds of things like measure gene expression by hybridization, by measuring the genotype of a person, by looking at hybridization with one version with a T versus the other version with a T, with a C.

and then sort of figuring out that I am a risk carrier for obesity based on these hybridization, differential hybridization in my genome that says, oh, you seem to only have this allele or you seem to have that allele. Microarrays can also be used to systematically synthesize small fragments of DNA. So you can basically synthesize these 150 nucleotide lone fragments across 450,000 spots at a time.

You can now take the result of that synthesis, which basically works through all of these sort of layers of adding one nucleotide at a time. You can basically just type it into your computer and order it. And you can basically order

10,000 or 100,000 of these small DNA segments at a time. And that's where awesome molecular biology comes in. You can basically take all these segments, have a common start and end barcode or sort of legator, just like pieces of a puzzle. You can make the same end piece and the same start piece for all of them.

And you can now use plasmids, which are these extra chromosomal small DNA circular segments.

that are basically inhabiting all our genomes. We basically have, you know, plasmids floating around. Bacteria use plasmids for transferring DNA, and that's where they put a lot of antibiotic resistance genes. So they can easily transfer them from one bacterium to the other. So one bacterium evolves a gene to be resistant to a particular antibiotic. It basically says to all its friends, hey, here's that sort of DNA piece. We can now co-opt these plasmids into human cells.