Being able to walk upright on two feet is a physical trait that distinguishes modern humans from our early ancestors. While the evolution of bipedalism has contributed to our success as a species, it has also limited the evolution of other features and increased our risk for certain diseases. Terence D. Capellini discusses the genetic research that is helping scientists better understand the relationship between bipedalism and our risk of developing knee osteoarthritis—a degenerative disease that afflicts at least 250 million people worldwide. By understanding the evolutionary history and genetics of this condition, preventive screenings and potential treatments may be developed.
Terence D. Capellini is Professor of Human Evolutionary Biology at Harvard University. He holds a PhD from the New York Consortium in Evolutionary Primatology (C.U.N.Y) working in the laboratory of Licia Selleri (Weill Cornell Medicine) and performed his post-doctoral research in the laboratory of David Kingsley (Stanford University). His interdisciplinary lab bridges functional genomics and genetics, developmental biology, medical genetics, and paleoanthropology. His lab currently focuses on how gene regulation shapes different bones of the skeleton, how interbreeding with Neandertals facilitated human skeletal adaptations, and most applicable, how alterations to gene regulation during human evolution have influenced the modern world-wide risk of joint-specific osteoarthritis.
Presented by the Peabody Museum of Archaeology & Ethnology, the Harvard Museum of Natural History, and the Harvard Museums of Science & Culture
Recorded March 2, 2023
Transcript
[00:00:08.08] Good afternoon, good evening. My name is Brenda Tindal. I am the Faculty of Arts and Sciences Chief Campus Curator and Executive Director of the Harvard Museums of Science and Culture. HMSC's mission is to foster curiosity and a spirit of discovery in visitors of all ages, enhancing public understanding of and appreciation for the natural world, science, and human cultures. This mission in mind, I am delighted to welcome you to tonight's lecture co-sponsored by the Peabody Museum of Archaeology and Ethnology and the Harvard Museum of Natural History.
[00:00:48.22] We are delighted to have Harvard professor Terence Capellini with us tonight to discuss his research on the evolutionary history and genetics of knee osteoarthritis, a degenerative disease that afflicts at least 250 million people worldwide. This is the first lecture in our spring program series, and it is indeed my pleasure to welcome both our in-person audience as well as those joining us via Zoom. To learn more about HMSC's upcoming lectures and events, I encourage you to visit our website at hmsc.harvard.edu.
[00:01:28.79] It is now my honor to turn the podium over to Dan Lieberman, Edwin M. Lerner Professor of Biological Sciences and Professor of Human Evolutionary Biology who will introduce tonight's distinguished speaker. Dan.
[00:01:44.49] So I'm really delighted and honored to introduce Terry Capellini. So Terry has been a professor in Human Evolutionary Biology the Department of Human Evolutionary Biology since 2013 after he finished a postdoc at Stanford in David Kingsley's lab. And before that, he did his PhD in New York at CUNY.
[00:02:04.98] And Terry was tenured about a year ago almost to the day, I think-- about a little over a year ago in HEB and for extremely good reason. In addition to knowing more about J.R.R. Tolkien than almost anybody I've ever met, Terry is by far-- and really. I mean, we know this for sure-- by far the world's leader in applying the field of evolutionary developmental biology, or known as evo-devo, to human evolution.
[00:02:30.64] So if you don't know what it is, evo-devo is the field that studies how evolution modifies development to generate change over time. It's a very exciting field, and it's important because evolution works through change, but that change has to have a developmental component, right?
[00:02:51.69] You can't-- so to put it crudely, you're different from a chimpanzee-- your ancestors looked like chimpanzees, by the way, if you didn't know. You're different from a chimpanzee because evolution modified the way in which chimpanzees developed and changed that to the way that you develop. And that helps you be the way you are.
[00:03:09.12] Now, figuring out how these shifts occur is really, really, really hard. Most of the researchers who study evo-devo do so with model organisms. They study animals in the lab. They can manipulate them. They can do all kinds of crazy horrible things to them. But obviously, we can't do that to chimpanzees or humans.
[00:03:27.40] And so what Terry has had to do, and what makes him world-famous and makes him the leader in this field, is he's figured out kind of a complex, integrative way of putting together different kinds of information to test hypotheses about changes in the evolutionary development of humans.
[00:03:43.11] So he combines genetic and genomic work with in vitro work with lab work on mice. He CRISPRizes-- if you know what CRISPR is. He changes mice into-- he puts human and chimp genes into mice and humanizes them in various ways. And it's super exciting, very complex, but extremely innovative work.
[00:04:05.11] He's done a lot of groundbreaking work on the evolution of human development, especially in regards to the skeletal system. And today, you're going to hear about a gene, GDF5, I suspect. You're going to hear a lot about GDF5 which Terry has made famous, or maybe it's made Terry famous or some combination of the two.
[00:04:22.02] And his work on GDF5 plus other genes and the regulatory effects of those genes have really revolutionized how we think about the evolution of bipedalism. That's the most important thing about being a human, really. That's what set us off on a different evolutionary path from chimpanzees.
[00:04:36.82] And as you'll see, these discoveries have yielded really exciting, important insights into disease, especially osteoarthritis, which is the most common disorder that causes disability in the world and really a major issue. So these evolutionary developmental implications are not only exciting for their own sake, but also because they have important clinical applications.
[00:05:03.16] And as you'll see, Terry is also a very gifted lecturer and teacher. Students love his classes. So you're in for a treat. And so without further ado, it's a real pleasure to introduce Dr. Terence Capellini to talk about when evolution hurts.
[00:05:17.28] Oh, excellent. OK. So thank you for having me. This is a really wonderful opportunity. I heard this is the first one of these this year. So I'm really great to be the inaugural speaker.
[00:05:27.99] So humans, as Dan mentioned, are quite different from other apes. So you can just compare humans to other apes like gorillas and chimpanzees, bonobos, and orangutans, you can see how very vastly different we are in our appearance, our phenotypic appearance.
[00:05:45.80] And if you take really any part of the body, or any part of the physiological system, humans are different from chimpanzees and other primates. We have, for instance, elongated and expanded our brain. We've changed our hand and foot morphology to be able to walk on two legs and to grasp objects and make tools and to throw. We've lost-- reduced body hair, and we've actually gained the ability to sweat.
[00:06:10.85] And if you look across just across the skeleton itself, the skeletal system, our bones, you can really see how drastically different we are from chimpanzees. And I'll talk a lot about these differences today.
[00:06:21.56] So we're quite derived. We're quite unique amongst primates. And with this uniqueness there also seems to be an ability to have unique diseases. So, for instance, we have diseases of affluence, like heart disease and diabetes and obesity because we have too much of things. We have diseases of old age because we live much longer than, say, chimpanzees who live to around 50. We live until at least 70 years old on average. That creates situations like arthritis and cancer risks and dementia.
[00:06:52.10] We have diseases of easy living. We process our foods a lot with tools, so we have dental crowding because we don't use our teeth the same way that we used to in the past. We get flatter feet, and we get increases in back pain because we sit a lot.
[00:07:04.83] And we also have diseases of altered environments. We have increased infectious disease risks. We get asthma because of pollutants. And we get increased skin cancer because we've changed the UV radiation coming into the planet. So we've really changed a lot of the environment. And as a result, we have this unique risk for disease as well.
[00:07:22.27] So are there any direct connections between how we evolved and these unique disease risks? And so my lab is really at that kind of interface. And I have a lot of wonderful lab members. Many of them are here in the audience. But they're also like-- they've graduated and gone to other better jobs. And over time they've studied lots of different questions in human evolutionary biology at this intersection.
[00:07:48.61] So, for instance, we study the effects of genetics on height and why we vary in body size and how this affects our skeletal disease risks. We study Neanderthals, our ancient human ancestors and how they've affected our immune systems. We study brain biology. We study our hands and feet and how they evolve to be different from each other.
[00:08:09.37] We study the shoulder and the pelvis-- these really wonderful pelvis bones that allow us to walk on two legs and give birth to a large fetal head. And we actually study knee biology and knee disease, which is a major topic for today's talk.
[00:08:21.72] The lenses that we use are many. We use a lot of different tools, and we integrate them to understand the biology of a trait. And the reason why we do this is because we have a simple model that we follow, or we follow this simple idea, which is when we come up with an idea, we have to be able to support that idea using multiple different types of data coming from different areas.
[00:08:46.76] And if, for some reason, we have a conflict where, for instance, a piece of data arises from some field that doesn't agree with our findings, we don't all of a sudden ignore that piece of data. We have to change our model. We have to incorporate it. So by using a holistic approach, by using all of these different areas of biology and beyond, we can address questions of human uniqueness with consilience, with internal agreement.
[00:09:13.11] So for tonight's talk, I want to mention that the major question I'm going to be asking is, How has evolution affected our hip and knees? And how has that contributed to our increased risk of disease, particularly osteoarthritis? Again, a question that's at the forefront of our lab that focuses on human uniqueness and disease.
[00:09:32.77] So walking on two legs arose pretty quickly after the last common ancestor of chimpanzees and humans. So between 6 and 8, 6 and 10, depending on who you ask, million years ago. And from this last common ancestor, we can see fossil species, such as Ardipithecus ramidus, showing evidence of some bipedal-like postures-- so particularly in their hips and their femurs or thigh bones. As you move down to their feet, they show a little less evidence of bipedalism.
[00:10:01.56] When you look at something like Lucy, the famous Lucy fossil from around 3.3 million years ago, you have a fossil specimen that actually has a really human-like hind limb, at least in terms of shapes, not necessarily in terms of size or length.
[00:10:16.93] Then you get to around 1.6 million years ago to a species within the genus Homo, which is our genus called Homo erectus. And these individuals actually had human-like body proportions. If you looked at a Neanderthal skeleton-- sorry-- a Homo erectus skeleton, it doesn't look too much different from a modern human skeleton in its biology.
[00:10:37.85] And so you can see pretty quickly that we've acquired the ability to be walking on two legs. And that ability eventually allowed us to move across the landscape, to traverse to different continents, to survive on different foods, increase cultural complexity and brain evolution, et cetera. So really, a hallmark trait for humans.
[00:10:58.22] So this evolutionary process rapidly shaped our knee biology. So if you compare-- this is a great ape knee compared to a modern human knee, you can see, for instance, that the femur, or the thighbone, this upper part is angled a bit.
[00:11:14.95] We have these surfaces for contact with the lower bone, the shin bone, which are stronger and larger because we transmit weight right down through our thighs to our shins as opposed to being bent. And we actually get a lot of bony support on our shin bone or tibia to support that increased weight. So these are really unique changes, and we actually have been able to discover that they arise developmentally even in utero.
[00:11:41.11] When we look at our pelvis, you can see even more drastic changes happening. So compare the chimpanzee pelvis to the human pelvis, you can very much see these massive changes happening. So in the chimpanzee, these blades called the ilia are very tall, and they actually sit on the back of the animal. So if I put my hands on your back like this, that's where they would be oriented.
[00:12:04.87] In humans, these blades have actually been rotated or curved in this direction. So they form or rotate this direction. And what that's done is allowed muscles that are normally on the back, like in chimpanzees, to now be on the sides, which helps us stand on one leg and walk on one leg-- important for movement.
[00:12:23.17] We've also changed the width of our birth canal. That's been changed at this region here called the pubis. We've elongated our-- enlarged our hip sockets to transmit more weight. And so there's been a massive number of changes happening to the pelvis in humans compared to chimpanzees.
[00:12:39.45] I would actually say that postcranial, or below the head, this is probably the most changed skeletal structure in the body. And it's really because it's allowing us to walk on two legs, but also give birth to a large fetal head, both essential for human existence.
[00:12:54.36] So what Dan mentioned very eloquently is that all traits arise through development. You have genes, and you have structures. And you can't just magically appear. They have to arise during the developmental period. And this developmental period could happen in utero, in gestation. It can also happen postnatally, and usually happens in combination.
[00:13:16.74] So this is just showing the stages of human embryonic development showing, for instance, the early single-cell zygote all the way up to an animal that looks like it has developing limbs, like four limbs and-- arms and legs-- to a point around eight weeks gestation where it looks more human-like in a lot of respects. It has really human-like legs and human-like arms.
[00:13:39.09] But during this process, what's happening is that you're not just getting morphogenesis or the appearance of form. What you're actually getting are a bunch of tissues that differentiate to form all the different structures in the human body, all the different tissue types in the human body.
[00:13:53.76] There are over 800 different cell types in the human body. They are rising developmentally all from a single-cell zygote. It's a complex, orchestrated process. It happens in all organisms. But some aspects of that process may be unique to humans.
[00:14:09.91] So the argument is, is that evolutionary changes to the development and how development happens is really what's causing the shape changes, the morphological changes, the anatomical changes that we see between humans and chimpanzees.
[00:14:23.85] So how has evolution then influenced how our knee and pelvis form, how they develop? So there's a big project out there called the Epigenomics Roadmap project that looks at human development. And they're able to study all different aspects of human development, whether it be brain, heart, lung, kidney.
[00:14:42.73] And they can discover the genes that are used in those cell types to actually look-- to actually orchestrate the development of the liver or the development of the heart or the development of lungs. They can also identify the regulatory regions-- the regions that control genes-- turn them on and off in different tissues.
[00:14:58.63] And at the time that the Roadmap project was happening, no one was studying the skeleton because it was actually very hard to study. You know the skeleton is a hard object. You know it survives in the ground after you die for a really long time. Fossils are basically mineralized copies of a skeleton.
[00:15:17.51] So the skeleton is a hard tissue. And to get cells from that tissue required a lot of technical achievements, which I'm very proud of my lab was able to do. And by having those technical achievements of extracting cartilage and bone cells from a developing skeleton, we can study it. It was a new window for us to study in human development. So we can actually start to compare our analysis on these data sets to other labs that are looking at the liver or the heart or the kidneys-- tissues that have been a little bit more easy to study.
[00:15:48.53] So during development of the skeleton at around this day, around 44 days in gestation, you get a model that forms of some of the bones. This is a model of cartilage. The cartilage is like-- you know when you're eating a chicken bone-- the end of the bone that's easy to eat, or you like to-- you can't eat the actual hard part of the chicken bone, but you can get that end. That's cartilage. And early in development, that cartilage is the whole model of the bone. It's not just at the ends.
[00:16:14.88] And so what happens is cartilage model forms. And eventually, it gets replaced by very hard bone. And we were really intrigued by this process because we never really looked to see what does the model of the human skeleton look like at the cartilaginous stage? So we actually did this study. And you can see by 8 weeks of development the human pelvis here in blue, which is staining the cartilage, looks like an adult human pelvis.
[00:16:42.62] For instance, the iliac blades, those blades I told you that are rotated around in this plane, are already rotated around. The hip sockets are large. The knee is expanded, and it has these extra surfaces that allow for contact and support.
[00:16:59.84] So by 8 weeks gestation, the cartilaginous skeleton, which is not bone yet, already shows the hallmark traits of being an evolved human biped, or walking on two legs. So we were excited by this finding. It means that we should be studying this period of development to understand how this happens.
[00:17:16.59] So we actually then were able to reconstruct the cartilage of the developing pelvis across six weeks to eight weeks of development. So this early stage of first trimester development where we can actually take out the cartilage and model it.
[00:17:32.81] And you can start seeing this is the femur or thighbone. This is the early pelvis. And then other parts of the pelvis form. Eventually, you get this pelvic blade. It gets bigger and wider.
[00:17:43.01] It starts to now curve in this direction in what they call the parasagittal plane. And eventually it forms as a cartilage model a unique human pelvis. And this is a direct window. This is two weeks of gestational development where this is all happening.
[00:17:57.42] We also did this for the femur, for the knee itself. And what you can see is the femur and the tibia start off as these like rudimentary structures that eventually take shape to form a femur bone and the knee joint proper with all the anatomical traits that we expect to be in the knee minus some that arise when we start to walk when we're children. So what this tells us is that evolution has targeted this early stage of cartilage development to make these models form the way they should.
[00:18:28.16] So what specifically then has evolution targeted developmentally to cause and orchestrate these cartilage changes, these changes in cartilage form that premodel the skeleton? So we need a little background to explore this.
[00:18:44.56] In every cell in the body, there is a region, the nucleus, that contains chromosomes. And chromosomes are basically bundles of DNA material, double helix material, what you've probably seen before-- the double helix-- the famous DNA double helix, which is really a bunch of bases that are linked to each other in the midline and then twisted. And these bases are the As, Gs, Ts, and Cs that you've probably heard about.
[00:19:11.03] And so what we know is that this long structure in one chromosome contains regions that are called genes and regions called switches. Switches are regulatory switches. And the analogy that we can talk about is house analogy, which I'll mention in a moment.
[00:19:29.47] So genes, just for a little background, are the pieces of the DNA that actually are used to make protein. So the different parts in your body, the different proteins that you have in your body that are coming from genes, the genes are giving the blueprint to make those proteins.
[00:19:45.02] You can think of a gene this way. A gene might be like the central heater in your house you have in the basement. Most of us, like myself, have it in the basement. And that central heater produces something, heat, that goes to every room of the house.
[00:20:00.23] So if there's a problem with your heater, it breaks, especially at bad times in the winter, the last time-- when you don't want it to happen, you lose heat throughout the house. That's bad. So genes have really essential functions across different parts of the body in the way that the heater has different functions across the house. And if you disrupt the gene, you can end up affecting the skeleton in every place where the skeleton forms. That's not good.
[00:20:29.29] Now, a switch is something different. A switch turns on a gene. You could think about a switch as a thermostat in each individual room of the house. That allows you to make the bedroom a little colder or warmer compared to the kitchen or the bathroom.
[00:20:41.67] And so if you, for instance, have a broken switch in the bedroom, well, guess what? You don't have heat in the bedroom. But guess what? You have heat in every other place in the house. So that's less damaging, but still not great.
[00:20:55.71] And you can imagine in the same context the developing skeleton has a gene that has different switches. Maybe there's a femur or a thigh switch, or there's a tibia a shin switch or a foot switch. And if you pick out that tibia switch, all of a sudden you just have short tibia, but the rest of your skeleton forms normally. So that's the analogy. Genes are the heating unit, and switches are the thermostats.
[00:21:21.64] So a single gene will often have multiple switches. And I kind of alluded to that in the previous slide. So imagine this gene is turned on in the embryo. This is a mouse embryo.
[00:21:32.81] And if you're not familiar with the mouse embryo, what you can see is this is developing head. Here's the eye. Here is a forelimb and a hind limb, and this is the tail. And anywhere you see blue in this is where you see the gene actually being turned on and used. So it's used in the brain. It's used in the limbs. And it's also used along the tail area.
[00:21:50.71] And what people have discovered is that there's separate switches that control this gene in very specific particular places. So, for instance, there's a switch that only controls the expression in the limbs. There's a switch that controls it in the tail and the axial system. And there's a switch that controls it in the brain.
[00:22:08.28] That's kind of cool. You can turn this gene on and off in different tissues without really affecting the expression in another tissue. So if I modulate this switch, I'll only affect it in the head, but I won't affect the expression in, for instance, the axial system or the limbs. So different switches allow for independent body parts to form independent of other parts.
[00:22:30.67] Now, a switch, though, is made up of genetic sequence. It's As, Ts, Gs, and Cs. And if you compare a human sequence of the switch to a chimpanzee sequence, you can see places where the base pairs are different.
[00:22:43.72] So here, for instance, humans have an A, and chimpanzees have A. That's the same. But over here there's a G, and the chimpanzees have a T. So the sequence itself is actually important for telling what the switch-- telling the switch what to do.
[00:22:57.99] And you can imagine that if I come into a bedroom and dial the switch, I might hold the switch differently than, say, another person who comes in and turns it rapidly. And you can imagine the sequence changes are really what's changing how fast or how slow that dial affects the thermostat.
[00:23:17.19] So a change like this could, for instance, lower the heat only a little or a lot in a particular room of the house. It doesn't kill the heat in the house. It doesn't kill the heat in the entire-- in the room itself. It could just lower it.
[00:23:33.91] And so, for instance, a tibia switch might have this sequence change in humans compared to chimpanzee, which makes the tibia just a little bit smaller, or a little larger, depending on the direction. So that's the analogy.
[00:23:45.67] Now, switches are unique to individual body parts. And therefore, when they have separate changes, they only affect that body part. So if I have a femur switch, and there's changes in humans compared to chimpanzees, it's only going to really affect the human development of the femur.
[00:24:01.42] And these switches can be found everywhere. So this is the genome-- the different chromosomes in the body. There's 22 autosomes and an x and a y. And if I show you the positions of switches, for instance, in the knee, all these red dashes are where those switches are in those chromosomes. And these individual switches could actually have different base pair changes between humans and chimps in many different locations.
[00:24:28.43] So building a femur, for instance, building a knee or building a pelvis requires lots of switches all of which, or many of which, can change in the sequence compared to humans and chimps. But finding these switches-- this is the critical part. Finding these switches is synonymous with finding a needle in a haystack. And there actually is a needle in this haystack. Does anybody see it? You see it. It's right there.
[00:24:56.99] I actually had it fainter. I made it brighter here just so you can see. It's really difficult to find these needles in this haystack, as it is to find the switches and the DNA changes that orchestrate why human knees are different from chimp knees, or why our brains are bigger than chimps.
[00:25:14.50] So our ongoing hypothesis, then, is that modifications to control of cartilage development during development underlie the real changes that we see in knee and pelvic evolution between humans and chimpanzees. And so what we need to do is develop an assay to find the switches and define the genes. And this is a tool kit under the area of functional genomics, understanding the functions of the genome. That's what that tool does.
[00:25:45.02] I'm not going to tell you about those tools. I can answer questions afterwards. They're complex. All you have to really know for this talk is that we can find the switches. We have a tool to do this. And so what we can do is take different parts of the skeleton, extract those cartilage cells, and then discover the switches. And we can do that at all different sites across the skeleton.
[00:26:07.85] What I'm showing you here is only the ilium and the femur and the tibia. I'm not showing you the other parts. But we've extracted all the different switches from different parts of the developing embryo.
[00:26:19.11] And what we can do, once we find these switches, is we can ask the very basic question. How many of these switches in the genome are found only in the distal femur, the upper part of your knee? And how many are only found in the upper part of your tibia or your shin? Or how many are shared between the two? And we can-- we have these methods that allow us to discover these site-specific and shared switches.
[00:26:47.63] So, for instance, for the knee, the regulatory elements or switches that are shared between this part of the knee and this part of the knee are about 502 in the genome that we were able to identify. Whereas, for the distal femur, the ones that are found right here, there's about 2,845. And for the proximal tibia or the shin, there's about 5,000 of them.
[00:27:08.03] So there's different switches that are unique to different body parts or shared. And now we've discovered these switches. So the next question then becomes, what can we do with them? How can we investigate sequences that make a difference in human biology?
[00:27:22.77] So has evolution targeted these switches to create the phenotypes that we see between humans and chimpanzees? So to do this, we use our next tool, which is comparative genomics-- comparing the genomes, the entire 3 billion base pairs in humans to an equivalent number in chimpanzees to find those needles in haystacks. But we filter using those switches. I'll show you how this works.
[00:27:47.85] So one of the types of changes that we're actually very interested in are changes where humans have changed DNA sequence compared to chimpanzees, gorillas, and other primates. And these places where there's conservation of genetic sequence compared to the changes in humans tell us that humans evolved sequence changes along the human lineage.
[00:28:15.77] Whereas, in the same region of the genome in chimpanzees or gorillas, there were no changes. And these places where humans have accumulated nucleotide or DNA changes are called human accelerated regions. We've accelerated the number of changes relative to other primates.
[00:28:33.56] There's not many of them in the genome. There's actually about 3,000 of them in the genome. So we can ask if evolution has shaped the knee or shaped the pelvis, and we have switches for the knee and the pelvis, do any of these switches have these places, these types of sequences that have acquired more changes along the human lineage that have been under what they call positive selection along human lineage compared to others?
[00:28:59.13] So we've done that. And this was worked on by Daniel Rokshar in the lab, who's in the audience. And so we've intersected these switches. And we've asked, Can we find these switches?
[00:29:10.11] One thing we wanted to do was ask if we found these switches that have these human accelerated regions, these regions that have changed in humans compared to other primates, do we see more of them than expected by chance?
[00:29:25.02] And we ask this question for a very basic reason. There's 3 billion base pairs in the human genome. By chance, if you took two different regions of the genome, or two different types of sequences, you might just hit some of those sequences.
[00:29:42.15] And so what we want to know is if we develop a background model of how much randomness there is in the genome, can we see that, for instance, our sequences, our switches have more of these intersections with these regions than expected by chance. And that's what this is showing.
[00:29:57.37] So the key point here is that anything above this line is a data set that shows more of these human accelerated regions intersecting with them than by chance. So other people have studied the human brain, and we know the human brain is very derived. Our human brains are four times the size of chimpanzees at least. And our brain size is very complex.
[00:30:23.08] And people have shown, a lot of people have shown, that if you look at switches in the brain, they have more of these accelerated regions than expected by chance. And so we did the same experiment because in science we want to recreate previous experiments.
[00:30:36.19] So we recreated this experiment. We took the brain we identified all the switches in the brain. And we asked, Do they overlap with these HARs, these Human Accelerated Regions? And, of course, they do.
[00:30:49.28] And then we did the other experiment where people have shown in the past that blood cells and the regulation of blood is not under this type of enrichment for accelerated regions. And we did that analysis. And we found that they fall below this line, so they're not enriched. So these are our positive and negative control experiments. They tell us that we're doing the right thing.
[00:31:09.62] So what about the switches that are found in different body parts? So here's a knee. So the switches that were actually found in the distal femur or thigh and the proximal tibia or shin that are common, that are shared, actually have less than expected by chance.
[00:31:26.47] They're really-- they're not enriched for these human accelerated regions. But the distal femur and the proximal tibia, the individual parts that drive knee development, they actually have more of these human accelerated sequences than expected by chance. So there's sequences in those switches that can make a difference.
[00:31:45.32] Then we ask the same thing for the pelvis. What's really interesting is that regulatory regions that are shared by this part of the pelvis and this and this and this and this, they are actually also enriched in human accelerated regions. And I just want to pause on that for a second because that's meaningful.
[00:32:01.90] The pelvis is a three-dimensional-- a complex three-dimensional shape. It allows us, for instance, to walk, but also gives birth to a large fetal head. As I mentioned, our head sizes are like three to four times the size of chimpanzees.
[00:32:13.42] We've got to pass-- females have to pass that head through the opening of the birth canal. So it's not surprising that the complex regulation of this structure that's shared across different tissues in the structure actually have an enrichment of these human accelerated regions.
[00:32:32.29] Then, of course, we found that the other parts of the pelvis, the individual control elements that control, for instance, only this part are also enriched. So what I'm trying to say here is that for the knee and the pelvis, we see evidence that the switches have been under evolution in humans, that they have more of these human accelerated sequences than expected by chance.
[00:32:54.31] So one of the things that Darwin was fascinated with was the idea that species evolve and split from one another from variation that's created within a species. I mean, Darwin was fascinated within species variation. He studied lots of different species, and he looked at variation within the species to understand what's going on in the species. Because the root of all variation that occurs between species comes from what happens within a species-- when the species start to divide.
[00:33:24.84] And so we wanted to know, were these knee switches that we found and these pelvis switches, what does genetic diversity look like in humans? All of us in this room are on Zoom. What does the diversity in these switches-- how does it differ from one person to the next?
[00:33:40.60] So we acquired a large data set called the 1000 Genomes data set. Thousands of people from around the world, all these different places where you see dots, and they've sequenced their entire genome. And so what we can do is ask, What does diversity look like in each of the switches? Is there a pattern?
[00:33:59.36] So if a knee sequence, for instance, looks like this, where all of these individuals have the exact same sequence, that probably means that that sequence is important. Because if a change occurred, it could alter the function. So, for instance, if an A occurred in this sequence, that might actually cause this individual to maybe not walk correctly or walk less correctly. And that might lower their fitness.
[00:34:26.65] And, as a result, that individual over time-- the sequence over time would be removed from a population. And this is called purifying selection or constraint. When you have a variant like this in a conserved region, it's not beneficial. It's harmful. So it'll be removed over time.
[00:34:43.70] On the other hand, you can imagine a scenario where the sequence varies very dramatically. There's changes across the sequence and across these individuals. This could tell us that this region of the genome is actually not under this type of constraint, that there's actually relaxation going on, that you can modify a change, make changes without having any severe consequence. So we call this a relaxation of selection or relaxation of constraint.
[00:35:10.05] So what we can do is actually go across the entire genome of these thousands of people and create a background model of what a variation looks like. So this is a background model of what variation looks like across thousands of people. It's just a normally distributed curve.
[00:35:31.48] And then we can say, well, if I only look at the genes, those heaters in the house, how much variation exists in the genes across all thousands of people? And you can see that the genes fall on the left side here. They're actually depleted of variation.
[00:35:51.36] Genes are used by many tissues, as I mentioned. If I remove a gene, I can maybe not form the entire skeleton, and that's bad. So a mutation that arises in a gene, a genetic variant that arises in the gene, could be damaging. So that's why it falls on the left side here.
[00:36:08.88] On the other hand, there are regions of the genome that change pretty dramatically from one person to the next because of viruses jumping into the genome and other things that happen that make the sequence more variable, very hyper variable. And we call those repeat regions.
[00:36:26.13] And everybody knows about these regions who studies genetics, and they fall on the right. They have more genetic variation than expected by chance. So those are the two outliers-- extreme constraint or extreme relaxation. So where do our knee sets fall? Where do our pelvic sets fall?
[00:36:44.79] So the regulatory regions that are common, the switches that are common to the pelvis or common to the knee, they fall smack dab in the middle. They're no different-- in us, there's a whole bunch of variation. It's not enriched or depleted. It's just the background model.
[00:37:02.76] But then you look at something like the proximal tibia-- so the upper part of the shin. And these series of switches, these 5,000 switches on average show less genetic variation. So in this room, our switches actually have less genetic variation compared to other parts of the genome.
[00:37:22.16] And the distal femur, the upper part of the knee, has less genetic variation in it than you would expect by chance. And if you sampled from the genome, you'd see less genetic variation in the distal femur and proximal tibia than anywhere else in the genome.
[00:37:37.26] And then, of course, the upper part of the pelvis, this blade that curves that I said was so important for curving, that also has less genetic variation across humans. So, in other words-- and that's compared to other parts of the pelvis.
[00:37:51.23] But there's an interesting observation here. The hip socket, the actual hip socket, where your femoral head goes right into your hip socket, the regulatory elements that control your hip actually have more variation than you expect by chance.
[00:38:06.30] So, on the one hand, we have parts of the knee and parts of the upper pelvis that have less variation than expected by chance. And here, we have more. And that likely reflects the actions of selection.
[00:38:18.28] So, for instance, this pattern here is evidence of constraint. The sequences are important. You cannot produce mutations in them. If you do, they have a deleterious effect, a damaging effect.
[00:38:28.65] On the other hand, you can go ahead and make mutations in the hip sockets which they have less of an effect on the development of the pelvis-- on the development of the hip. So that's how we start to think about these switches in terms of genetic variation.
[00:38:42.83] What's really interesting, we've been able to do this for the pelvis working with wonderful collaborators from around the world. We've taken the pelvis, the actual physical bony pelvis, and we've measured a whole bunch of different metrics across the pelvis. In this case, we're looking at the ilium-- the width of the ilium. That's just one measure.
[00:39:03.29] And we start to say, How much variation is there in people around the world? And so these are different populations. Each one is represented by 30 to 100 different people. And you can kind of see that across populations, there's not a lot of variation.
[00:39:19.19] You don't see a big mountainscape with one population showing lots of variation and another population showing less. It's pretty consistent. And actually, it's pretty low. This is a low amount of variation. So that for that ilium, the measurement we took on the ilium itself, it doesn't vary that much across human populations, nor even within a population.
[00:39:39.20] If, on the other hand, we look at something like the height of the hip socket, we actually see that it varies dramatically from one population to the next. And these error bars within the population are quite large, meaning that individuals within the population have a lot of variation, too.
[00:39:55.10] So just to back up one slide, remember this pattern. The hip socket switches-- the switches that make the hip socket form-- had a lot of variation. And across populations, there's a lot of variation in the morphology of that hip. And there's less variation in the knee switches-- sorry-- in the ilium switches. And there's actually very little variation in the ilium morphology.
[00:40:19.46] So, in other words, if we can look at the genome and find all the switches, the patterns of variation we see in the genome across humans kind of matches what we see in the morphology, in the phenotype. And this is across the entire genome.
[00:40:33.07] So this kind of pattern that we're seeing where we see knees under constraint and hips allowed to kind of vary, what does that mean for our risk for disease? That's where I was going with this talk. What are the impacts of this ancient evolutionary process on our modern health?
[00:40:51.63] So, as Dan mentioned, osteoarthritis is the leading cause of disability-- one of the leading causes of disability in the world. It's anticipated by 2040, around 40% of people over the age of 65 will have osteoarthritis of the knee. That's at age 65. It's going to be much higher by the time of 80.
[00:41:10.89] Having osteoarthritis of knee is debilitating. It means you're not physically active, and it has a lot of comorbidities associated with it, like heart disease and diabetes. So it's a really big risk factor for dying early.
[00:41:25.20] And it's a unique degenerative disease. It actually-- the cartilage that survives later in life that lines the joints, the same cartilage that kind of helped form the model in the first place, that cartilage deteriorates. So this is a normal healthy hip, and this is an osteoarthritic hip. We probably all have relatives who have hip replacements or knee replacements. This is what some of their hips look like.
[00:41:48.31] And this is a knee, for instance-- a normal healthy knee, and this is a degenerative knee. So knee osteoarthritis is a degenerative cartilage disease. Of course, there's other inputs that cause knee osteoarthritis, but it's mainly a cartilage disease. So what's really interesting is if you study chimpanzees in the wild or in zoos or gorillas or orangutans, they don't have much osteoarthritis. They have very low rates of osteoarthritis.
[00:42:13.91] So our prevalence of osteoarthritis in human populations is much higher. It's actually a unique kind of feature of our disease pathology in humans compared to something like a great ape. So it's derived. In many ways, our skeleton is derived, but our risk for osteoarthritis is also derived. It is much higher than what you see in other primates.
[00:42:33.59] So what we wanted to do was ask if there has been evolution on these knee switches to shape our knees, to shape our pelves and our hip sockets, do they show evidence of conferring some risk for this disease, osteoarthritis? So what we did was we found all the genomic regions in the genome that confer risk for osteoarthritis. And this is done by other people. They're called genome-wide association studies.
[00:43:01.02] I won't go into the details. They're just-- they're methods that allow an individual researcher to find places in the genome that associate with risk-- increased risk for a disease. In this case, osteoarthritis. We found about-- there's about 100 of them out there in the genome. And these are risk alleles.
[00:43:18.59] Like, you've probably heard that some people have a breast cancer gene. Well, imagine you have a breast cancer-- an osteoarthritis mutation that increases your risk. That's a very similar way to think about it.
[00:43:29.43] So we compiled these regions of the genome, and then we asked, Do they fall within our knee [AUDIO OUT] at a greater rate than expected by chance? In the same way we asked about the human accelerated sequences-- Do we have more evolution in these sequences? We're asking, Do we have more disease risk in these sequences?
[00:43:48.64] So this is a background model. This significant line, again, tells us what can be expected by chance. Anything above this line is more significant. Anything below this line is less significant.
[00:44:00.40] So what we can find is that knee switches, the switches that turn on the knee during development, actually have more osteoarthritic risk variants than expected by chance. That's very interesting. These are switches from the embryo that help build the knee, but they confer risk later in life. These are switches that showed constraint. They weren't tolerating genetic variation. But when the variation does arise in them, they cause OA later in life.
[00:44:31.41] The pelvis generally didn't show that pattern. The hip socket kind of showed a trend toward significance. It didn't reach it. And that's probably because we don't have a lot of data yet on the genetics of hip OA-- osteoarthritis. We have more on knee osteoarthritis.
[00:44:46.03] So this pattern might actually change once we get more and more regions of the genome that we can identify as affecting hip osteoarthritis. So what this is saying is that the regulatory regions, the switches that turn genes on and off in the knee, actually have a conferred risk for osteoarthritis.
[00:45:03.28] Now, the next thing we wanted to do was say, What happens if we actually look at patients? What if we take a bunch of patients that have osteoarthritis-- do they have more osteoarthritis variants-- disease-causing variants-- in these switches?
[00:45:21.30] There's a big study called the Osteoarthritis Initiative that looks at like 4,000 to 8,000 people, and they have some genomes for them. And they have MRI data from the knee to look at the extent of osteoarthritis.
[00:45:33.75] And so what we asked was, If we take all the switches for the knee, do we see more osteoarthritis risk variants in these patients than nonpatients, than controls? And what we found was that that was the case, that patients here have more mutations in their switches than nonpatients-- controls.
[00:45:58.20] Now, one could simply ask a very basic question. Maybe these individuals have more mutations in their whole genome? And, as a result, because they have more mutations, they just happen to have more mutations in their knee switches than controls.
[00:46:12.60] So we did the same thing, but this time took regions that are in blood-- regulatory switches that are in blood. And if it's the case that OA patients have more variants genome-wide, then we should see in blood regulatory regions the same pattern. And that's not what we saw. We actually saw that in the blood switches, humans, human patients for OA, and nonpatients, controls, didn't show a difference.
[00:46:36.22] So there's a burden of having mutations in these switches that confers an increased risk of osteoarthritis. And what we've developed is a model that discusses this.
[00:46:48.56] So ancient selection-- being able-- walking on two legs, the need to walk on two legs, the evolutionary process of forming bipedal-like structure has shaped our knees. It's caused some sequences to go under acceleration increasing the changes in nucleotides to cause us to be able to develop a knee that allows us to walk.
[00:47:10.49] And in modern populations, there's a constraint on that process. You've built a knee that works. Don't change it. So don't make any changes to your knee because if you do, there can be ramifications.
[00:47:22.67] Of course, if you change your knee too much developmentally, you might not be able to walk properly when you're born. But that's not the type of changes that we're seeing. What we're seeing are small changes-- changes that accumulate, that may not affect really how you walk right now.
[00:47:39.53] But when you get older, and maybe you lose muscular skeletal coordination because you get older and you can't walk correctly, then you start seeing a conferred risk of osteoarthritis. These mutations that subtly affected shape, maybe they don't affect shape anymore in the same way. Maybe they start to cause disease. And that's the model that we have, that genetic variants on the backdrop of this constrained process to build a knee increases inherently our risk for osteoarthritis.
[00:48:08.11] And this is a really important part of this talk. When we think about evolution, we think about natural selection. We think about adaptation. We don't often think about what the costs are.
[00:48:19.18] If you build something with a certain material, that material allows whatever you're building to work correctly in the context of which you built it. But if you change the context, that material might not be beneficial to the functioning of that piece-- of that item that you've built. And that's the way evolution works.
[00:48:38.87] So the real question then is we've identified these variants. We can see that patients have more of them than nonpatients. They're in knee switches that have under selection and constraint. Do they actually cause osteoarthritis?
[00:48:53.26] And so one of the genes that we've been studying in the lab is this gene called GDF5-- the Growth Differentiation Factor 5. Long name, it's involved in [AUDIO OUT] the joint. When I want to build the elbow joint or the wrist joint or the hip or the knee, this gene gets turned on and tells the cells, hey, start building a joint.
[00:49:14.03] And this gene is one of the leading risk factors for osteoarthritis. And the mutations that cause, or could cause, osteoarthritis occur over a very large gene region. So this is-- the gene itself-- this is just a depiction of the gene on the DNA. This is the gene. This is another gene. And each of these marks is a separate change-- a T to an A, or a G to a C. And we don't know which ones cause osteoarthritis. So we have to use our switches to help us figure this out.
[00:49:48.10] So what we did was we took switches from each of the tissues that are described. Each of these boxes is a position in the genome where there's a switch. So this is the region of the genome for GDF5 and the [INAUDIBLE] gene. And each black box tells you, hey, there's a switch.
[00:50:04.12] So in the hip, also called the acetabulum, each of these boxes shows the location of a hip switch. And this is the proximal femur-- upper part of the upper thigh. This is the distal femur-- lower part of the thigh-- proximal tibia-- upper part of the shin of the knee.
[00:50:19.65] And what you can see is that there's a bunch of switches, but only really one of them right here overlaps a knee switch. And this switch actually has a mutation that goes from C in controls to T in patients. Does this cause osteoarthritis? Does this single change cause osteoarthritis?
[00:50:39.10] So we made a mouse. This is where we do CRISPRizing in mice. And we went into the mouse genome, and we made a single change, a single base pair change. We edited the C to a T. We went from the nonrisk to the risk allele. And then we can produce mice that have two copies of the C-- one each parent.
[00:50:57.92] The offspring has a C and a C, one from mom and one from dad, or a C and a T, or a T and a T. And we ask, Do they get shape changes to their knee, and Do they get osteoarthritis?
[00:51:07.99] And this is what we found. And what you're seeing here are each of the parts of the pelvis or the femur or thighbone-- this is the tibia or shinbone. And what you're seeing is the shape change between the mice that have the nonrisk change versus the mice that have the two copies of the risk variant. In other words, it's just superimposed.
[00:51:32.73] Anywhere you see red or blue is where you see a shape change between the affected individual with the T versus the nonaffected. And so you can clearly see that the T changes the morphology or shape of the femur, or of the top of the tibia where the knee joint sits. So the single base pair change caused the knees to now look slightly different from a normal setting.
[00:51:59.73] These mice are normal. You look at them, they run around the cage. They're perfectly fine. But then you start to look at when they're one year old, and they start to get osteoarthritis. So we have a score that we can measure osteoarthritis. And each of these black dots is a different mouse.
[00:52:14.61] So in a normal mouse, this is what the knee joint looks like. This is the lower part of the femur. This is the upper part of the tibia. This is like the space where your meniscus sits, where your pads are that protect your knees.
[00:52:28.86] And this is a mouse that has osteoarthritis where the cartilage, this red area and cartilage, is ripped and gone and destroyed. This is what a human knee could look like with osteoarthritis, where you see cartilage tears and lesions. So these switches, they actually do confer osteoarthritis when they have the mutations in them. And this was proof of principle that this actually can cause osteoarthritis, a complex disease phenotype.
[00:52:55.01] So let me summarize the talk tonight. Evolution has targeted our knee and pelvis by altering how they form, how they develop in utero. This has been beneficial to our survival, our ability to walk on two legs, and even to run. Dan studies running, so threw that in.
[00:53:13.26] This causes subtle shape effects on our knees when mutations arise in them. But those effects are not really going to impact when we're young. But as we get older, and as we start losing musculoskeletal coordination, and as we have increased inflammation in our bodies, as we age, we get an increased osteoarthritis from those same variants that have been modified.
[00:53:36.09] And we can use the whole technique that I showed today to really pinpoint these mutations, like we did a GDF5, and show that they can cause osteoarthritis. And if we can do that, what we can start to do next is figure out how to therapeutically treat how switches act in the knee, and how they start to help form and maintain a healthy knee.
[00:53:58.11] So with that, I want to thank members of my lab who have gone on to better things, and members who are there who have worked on this project. I want to thank my collaborators. This beautiful imaging is done by a colleague at Boston Children's Hospital named Ata Kiapour; all my colleagues who worked on the pelvis project.
[00:54:14.88] I've received lots of funding from different sources, and I've worked with lots of different cores and companies to help do this work. Thank you for attending.
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