University Place

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Cassie Immel

Thank you for watching Wednesday Nite at the Lab tonight. This evening we have Dr. Farrell, who's served as the dean at the University of Wisconsin Medical School from 1984 through July 2006. One of his many accomplishments includes working towards a transformation of the school to an integrated School of Medicine and Public Health, and he continues to serve the SMPH through numerous leadership, funding, and educational activities. To kind of talk about a few of his current activities, right now he's the principal investigator for the Wisconsin Cystic Fibrosis Neonatal Screening Project. He's also leading national research efforts devoted to quality improvement in the apparition of cystic fibrosis newborn screening programs. And without further ado, I would like to welcome Dr. Farrell here to talk about cystic fibrosis. Thank you.

APPLAUSE

Cassie Immel

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Phillip Farrell

Well, thank you very much. I really appreciate the opportunity to visit with you tonight and summarize a project I've been working on for several years. And I'll tell you why I got into it and also explain why I have these bones here and teeth. I'll tell you what they are in just a second. I'm going to give you an overview of the project, sort of a limited amount of information on the methods and the biotechnology involved. My collaborators, particularly Cedric Lamarche who's back in Brest, France, in Brittany, have really been very, very important in this project in developing some excellent methods. I've also worked with Martin Shafer at the Wisconsin State Laboratory of Hygiene on some very advanced methods of trace element analysis using cores from femurs. And there's a number of other major techniques involved. Radiocarbon dating, for example, I'll tell you more about my collaborators in a minute. I'm going to really focus on a passion I've developed, namely to understand the ancient origin of cystic fibrosis. I'm going to explore this in archaeological genetic studies that are unique and associated studies, such as radiocarbon dating and trace element analysis, and ultimately what we would like to understand is the presumed heterozygote advantage for individuals who have one mutation in the CFTR gene and, therefore, are heterozygote carriers of this disease. And about one in 30 Europeans and Euro-Americans are carriers of a CFTR mutation. And that means in this room there are two or three individuals that are CF heterozygote carriers as they have at least one mutation in the CFTR gene. I'm very interested in this because of my interest in cystic fibrosis, epidemiology, and newborn screening for CF, and I'll explain that to you in a few minutes. So what are these bones and teeth? Well, this is femur, as you can see. This would be the knee joint here. And there's the head of the femur. And this bone is about 1900 years old. It was from a burial outside of the original walls of Londinium, which is a Roman city now known as London, and this is from a Roman citizen that was buried about 100 AD. And all these holes are here because we took cores, we drill out cores for radiocarbon dating and trace element analysis. This is another one about the same age. You can see in this case the knee joint is broken off. The distal end, the far end, is broken, and there's some contamination with soil here. And there's the head of the femur. This is a tooth from a similar, these are obviously from the Roman period, occupation of London. But this pre-Roman. This is from an Iron Age burial about 300 or 400 BC outside of Verona, which was a Celtic city occupied by individuals who traveled from an area near Vienna through the Alps and to northeastern Italy to settle that area before it was conquered by the Romans. And here's a tooth from the natural history museum in Vienna from a burial about 400 BC. Iron Age Celtic burial again. And we use these teeth for extraction of ancient DNA and analysis by molecular genetics methods. We have teeth that are as old as 4,000 years old from the Bronze Age that we've analyzed from a cemetery to the northwest of Vienna along the Danube River. And I'll tell you more about that in a few minutes. So let me proceed now. I want to acknowledge all my collaborators. This looks like a big project in terms of the number of people involved, and I guess it has become a big project. So here at UW Madison, Martin Shafer, who's probably the world's most expert analytical chemist for trace elements. This Inserm unit is a research unit in Brest, France. And all these individuals have worked on it. Several people here at UW Madison. Tom Stafford from the Stafford Research Lab doing radiocarbon dating. Probably the world's foremost European archaeologist, Barry Cunliffe, or I should say Sir Barrington Cunliffe because he's been knighted, from Oxford University. And from the British Museum, another archaeologist, another archeologist from the Museum of London, who told me to take these specimens back to Madison, these femurs. We were just going to take the cores in the basement of the Museum of London, but he said no, take the whole ball, we're not going to use these for anything. We had to bring them through Gatwick Airport and then through Cleveland. It wasn't easy but we got it done. Normally, we would have shipped them back but he just told us the last day we were there just take them back with you. We just didn't have time to work on a shipping plan. And then Maria Teschler-Nicola who's an anthropologist and archaeologist along with Peter Ramsel at the University of Vienna. So these are some of our collaborators. Now I want to tell you a little bit about cystic fibrosis so you understand the disease. It's a genetic disorder autosomal recessive. So both parents have to have one cystic fibrosis mutation in order for their child to have cystic fibrosis. You have the disease if you have two cystic fibrosis mutations. The incidence of the disease varies depending upon the ethnic or genetic background. So among white Americans, one in every 3,000 babies born in this country will have cystic fibrosis. And Hispanic Americans about one in 6,000. I'm having trouble keeping this mouse going. Okay, there we go. One in 6,000. African Americans about one in 10,000. And if you run the numbers, you find that about one in 30 are heterozygote carriers among white Americans. The disease is manifested by gastrointestinal abnormalities due to pancreatic insufficiency, inadequate function of the pancreas, which is a digestive organ, and that causes intestinal malabsorption and malnutrition. Secondly, chronic lung disease, which is potentially fatal. And then, finally, the third principal manifestation is a high concentration of salt in the sweat. And that's the traditional diagnostic test for cystic fibrosis. We know from, I should tell you, this is kind of a peculiar pattern of symptoms that you would have the sweat grands involved, you would have the lungs involved, and you would have the gastrointestinal system involved. It's difficult to understand, or it was prior to 1989, why these three organ systems were involved and why there's other clinical manifestations, but a major breakthrough occurred in 1989, published in Science on the 8th of September, 1989, discovery of the principal cystic fibrosis mutation, which is known as the delta F508 mutation or the F508 del mutation. And that was actually the discovery of the gene by discovering the principal mutation. 70% of cystic fibrosis chromosomes have this mutation. It's an interesting mutation. It's a three base pair deletion at codon 508. And when that mutation occurs you get a mutant protein. And incidentally, the CFTR protein that comes from the CFTR genes is a chloride transfer protein that embeds itself in membranes like the epithelial cells, the lining cells of the sweat gland ductuals, and what this protein does is simply facilitate the transport of chloride ions and, secondarily, sodium ions. So at the 508th codon, if there's a three base pair deletion, you get a mutant protein that's missing a phenylalanine residue or one of the amino acids in this protein which has 1480 amino acids. Now, this simple mutation causes some major problems, and this figure shows an incidence of one in 1,000, which is true in Ireland, to one in 5,000 among Caucasians. One in 5,000, which is what you'd find in the south of France, for example. So the CFTR gene encodes a chloride channel protein and the abnormality is defective chloride transport which causes these abnormalities in the lungs and the pancreas, etc. So quite a number of organ systems can be involved, and you'd think this would be a very easy disease to diagnose, but if you look at the age of diagnosis among CF patients in the United States, this is 21,588 of the roughly 30,000 CF patients in the United States, what you find is that about two-thirds of the patients are diagnosed in the first year of life but about one-third have a long delay in their diagnosis and many of these patients are really quite ill by the time they're diagnosed, surprisingly. And the other thing I should mention is that we know that 5% or 10% of children with cystic fibrosis can actually die undiagnosed by the traditional method of diagnosis. And we also know that females, girls with CF are diagnosed at an older age than boys. And that's the reason why we got interested in assured early diagnosis through newborn screening. So we've worked on this with an NIH grant since 1984 year at the University of Wisconsin in collaboration with the Wisconsin State Laboratory of Hygiene, which is just next door. I guess I should say just up the street. And this newborn screening for cystic fibrosis involves DNA analysis or molecular testing. I won't go into the details of that. And the test we developed here on the Madison campus is now used around the world so that in the year 2010 about eight million babies were screened for cystic fibrosis using a newborn screening method, all of these regions that are colored in green, I mean in red. So this is the United States, many of the provinces of Canada. I want to tell you why Ireland is green here. I spent quite a bit of time in Ireland, where my ancestors are from, because Ireland has the highest incidence in the world of cystic fibrosis, and they wanted to get into newborn screening but they really needed some advice and they needed some political help. I wound up meeting with the prime minister pushing this along. But I showed this slide with Ireland white. Northern Ireland has been screening for a while as part of the UK system, but I promised them that if they got a program underway I would color Ireland green on this slide.

LAUGHTER

Phillip Farrell

And I told them I was going to try to get 40 shades of green because of the song "40 Shades of Green" but decided to go with one shade of green. Newborn screening actually began in New Zealand and Australia, but we got involved quite early here in Wisconsin because we're very concerned about the poor condition of children being diagnosed in this state because of the delay in diagnosis, and we thought there would be a better way, namely early diagnosis through newborn screening. That's what we got into. But when you do a newborn screening test or any kind of screening test you get false positives. Not every test that's positive means that the baby has cystic fibrosis. And, of course, the same is true for any kind of screening like screening mammography. About 7% of positive mammograms are false positives. That is, the woman does not have cancer but had something detected. It's about the same for PSA screening in men for prostate cancer. About 10% of the positive tests are false positives. In other words, the PSA is high but the man does not have prostate cancer. And newborn screening is somewhat similar. It's set up that way because what you don't want is a false negative. You don't want a mammogram in a woman that has breast cancer to be a negative mammogram. And you don't want a PSA level in a man that has prostate cancer to be normal. And so you set the cut off point so that you accept false positives in an effort to try to avoid false negatives. So with regard to newborn screening, about 5% of the newborn screening tests that we do, and we do 29 newborn screening tests, in other words, we take a small sample of blood from a baby, the so-called PKU test, we actually do 29 tests, but about 5% of these result in a false positive test. It's about 200,000 families per year in the United States, and only.1% result in a true positive that is a baby that has a congenital disorder. So that's about 4,000 infants a year. Explaining these false positive tests is difficult. And especially when a woman has just delivered a baby, this is not a good time to have a conversation about something like that. And so we'd like to know more about the causes of false positives and what we can do about it. In the case of cystic fibrosis, there's about 10,000 false positive tests per year in the United States and about 1,000 true positives, that is babies diagnosed with CF. >> If you did a repeat test would it still be a false positive? >> Yes. I'm talking about tests that wound up being repeated. In other words, the initial was positive, the second one was positive, and then after a positive screening test a baby needs to come for a diagnostic test, a follow-up test. And what I'm saying is those follow-up tests would be negative. So in the case of cystic fibrosis, here's the typical thing. We detect one mutation and the baby comes in for the diagnostic test, which is the sweat test, and the sweat test is normal. So we know that baby is the CF heterozygote carrier because the baby does not have cystic fibrosis because the diagnostic test was normal, but we detected by our DNA testing one mutation. So we know that that baby is a CF heterozygote carrier. And parents have been asking me and my colleagues for a couple of decades what does that mean for my baby? My baby has one mutation, does that mean my baby is going to have a little bit of cystic fibrosis or have some kind of a problem? And we don't have an answer to that question. As a matter of fact, we don't have answers in general for genetic disorders that are metabolic in nature, like PKU, about what the situation is for the heterozygote carrier with one copy of the mutation with one exception and that's sickle cell disease. And you probably have all heard that, thanks to Linus Pauling, it was determined many, many years ago that sickle hemoglobin carriers, that is they have one copy of the sickle hemoglobin gene, are resistant to malaria. And what happened in Africa, particularly northwestern Africa, is the severe and potentially fatal malaria there does not affect those that are carriers of sickle hemoglobin. And so over the centuries, that population has been enriched in individuals that have sickle hemoglobin one mutation and they're carriers of sickle cell disease. Now, the children who would have been born from two individuals that were carriers of sickle cell disease that had sickle cell anemia, over the centuries probably would have died young. They live now because they get good treatment. But the sickle cell hemoglobin carriers had a resistance to malaria, and so there is what we'd call in the Darwinian terminology a selective advantage for carriers of sickle hemoglobin. You understand that? A selective advantage, they were selected for because of the endemic disease malaria there. So something like that must be the explanation in the case of cystic fibrosis. There's some selective advantage and that's what we've predicated our project on. We believe that selective advantage goes back to prehistoric times for reasons that I'll explain. How far back? We think it's back somewhere around 2500 BC. We think that people of the so-called Celtic culture that were great travelers migrating around Europe probably were involved in spreading this mutation. And so we think in the European Bronze Age and European Iron Age the population became enriched for some reason in this delta F508 mutation. And we've looked at some modern data that I'll show you that help us understand that. But we're trying to find out why the frequency of this mutation is so common that one out of 30 Europeans and Euro-Americans that are Caucasian have this. So we looked at the distribution in Europe, the haplotypes, that is the ancestral background, genetic background. We've looked at some indirect evidence, and we did some work with animal models. In fact, that's how I started out, studying transgenic mice that had one copy of the delta F508 mutation. And I did some research to show that they might be resistant to heavy metal toxicity, particularly lead toxicity. And so that's how we sort of proceeded with this project. Now, there has been a dispute in the literature as to the age of the delta F508 mutation. One of the articles suggests that it goes back to 50,000 BC. So a long way back. Another article suggests somewhere around 1,000 to 4,000 BC. And then my colleagues in Brittany have developed a method with modern DNA to suggest that mutation goes back to around 1,000 or 2,000 BC. I'll show you those data. Why are we so interested in when this mutation arose? It's because if you know when then you can get some kind of an idea or at least develop some hypotheses about why. Why there was a selection process for this mutation. And when you do that, you can want to look at endemic risk factors like the presence of endemic malaria or something else, environmental exposure, some kind of a nutritional stress, some kind of an infection. So we've been using direct evidence from skeletons we've studied like what I've shown you here, and we've also been looking at indirect approaches with modern DNA. These are some of the speculations, sorry about the misspelling there, or hypotheses to try to explain the CF heterozygote advantage. Protection from cholera, tuberculosis, for which there's some evidence but it's very limited and I don't think it's correct, other infectious diseases, syphilis, influenza, plague, protection from diseases of the lung like asthma, cancer, infertility, and we've added to the list metallic toxicity. So these are all things that could, in theory, explain why there's this selective advantage for the CF heterozygote carrier. When you try to organize a project like this you can sort of take advantage of what we learned about sickle hemoglobin and malaria and try to develop evidence criteria for selective agents. The best, I believe, that's why I've highlighted this orange, is a molecular or cellular strategy which we're now moving towards to develop a plausible mechanism or explanation by which being a heterozygote provides resistance to a significant stress over time. There should be a clinical correlation between the host and their genotype, their genetic makeup and the disease mortality or morbidity. A geographical relationship would be very, very useful. That's why in northern, northwestern Africa the endemic presence of a very severe form of malaria and the enrichment of the population in sickle cell carriers correlated so well this spatial distribution. You like that to correlate well for both a high incidence and a low incidence. That would be ideal. And then historical or temporal kind of evidence that a long-standing presence of the agent over time correlating with the incidence of the disease. These are kinds of evidence you try to gather in this type of a project. So these are the key questions we're looking at to try to get at this probable delta F508 selective advantage. So when did the mutation first appear, using indirect and direct methods? Where did this mutation originate? We think, oops, we think that it originated somewhere in northwestern Europe up towards the Scandinavian countries. We think the bulls eye here is Denmark. In other words, we think that there was back in the Bronze Age, about 2,000 BC, an Adam or an Eve with the delta F508 mutation, that the mutation arose way back then. And we're basing that on the fact that the delta F508 mutation is most prevalent and frequent here and as you get farther and farther from there, you see a decrease among cystic fibrosis patients and the percent with delta F508, looking at these modern delta F508 percentages. So to get at this where, we've also sort of thought about how people migrated out of Africa. And I just want to take a few minutes to review that, although most of you probably already know that. So we're interested in why there's a selective advantage and testing out different possibilities like tuberculosis or metal toxicity. And then we're interested in how this genotype persists, why the carriers persist. So, this comes from National Geographic, and as you, I'm sure most of you know, we're all Africans originally. Modern humans, Homo sapiens, developed, originally, in Africa. And we know that that was probably for modern humans, Homo sapiens, about 200,000 years ago. And that some time around 50,000, 70,000, maybe 90,000 years ago, these Africans then crossed at the tip of the Red Sea at the southern end of the Red Sea where it's narrow and they crossed over the Red Sea into Yemen, present Yemen. And they were then living in the Arabian peninsula so you could say all of us are really African Americans and we're also Arabian Americans because that's where we all came from. And in what must be considered the greatest travel of all time, even greater than the travel to the moon, modern humans then moved out from the Arabian peninsula to the Middle East. By 50,000 years ago they already were in Australia and somewhere, and they migrated into Europe around 30,000 years ago. And they also migrated across the Bering Straits into North America somewhere probably around 15,000 years ago. And so why am I bringing this up and why is this important? Well, if Europe was not settled until, by modern humans, until something like 30,000 years ago, it would be very interesting if the CF mutation is really, the delta F508 mutation is really 50,000 years old because you would have expected that you might find cystic fibrosis in India and in Asia and you might find it in the aboriginal Australia, etc, etc, but you don't. Where you find it is in Europeans and individuals who migrated from Europe to North America to Australia to New Zealand. And that's one reason why we think the mutation is younger than that period of time. So that's why I say you have to have some knowledge of history and how humans migrated to understand the ancient origin of certain genetic diseases. So, let me tell you why we think it arose in the Bronze Age and that there was an Adam or an Eve with cystic fibrosis in northwestern Europe during the Bronze Age. So we did some studies taking advantage of the methods that were developed in Brittany with modern DNA. What we call family studies or trio studies. So we obtained blood specimens from CF patients that were homozygous from the delta F508 mutation. That is to say they had two copies of the delta F508 mutation. And we also obtained blood from their mother and from their father, and we showed that the mothers and the fathers were delta F508 carriers. And we did this from three regions of Europe with relatively high incidence of cystic fibrosis. 27 trios from Austria, and these were native Austrians living in Vienna, 16 from Brittany, near Brest, France, and 20 from Ireland and we extracted the DNA for molecular genetic analysis in Brest. And I won't go into the details on the methods but we looked at regions of DNA around the CFTR gene and we determined what their haplotypes were, that is what's their ancestral genetic background, and then using a very interesting program developed in Paris called the ESTIAGE program for estimating age, we determined, we estimated, how many generations back these individuals had a common single ancestor. So you see what I mean? We've got DNA from all these individuals that I told you about, and we look at what their genetic variation is, haplotypes, and then we go back by this computer program to when they had a single common ancestor, we estimate that. And this is what we found with 95% confidence. That the Austrians, converting generations, 25 per generation to years, the Austrians, the age of delta F508 mutation is about 3,650 years ago, 1650 BC with a fairly wide range. Brittany, almost identical. Ireland, also very similar. So all of these are Bronze Age. That is, they're somewhere around 1500 to 2,000 BC. And that's why we think that most likely that's when this mutation arose. And that's, we think, the answer to when. Now when you know when, then you can start thinking about what happened during the Bronze Age and after the Bronze Age. How did the ancient Europeans migrate through continental Europe and the Atlantic islands, Britain and Ireland, for example, off the coast? You can also think about what kind of stresses did they have during that period of time and beyond? What sort of endemic infectious diseases were around? What else were they exposed to? It gives you an ability to sort of think through what might have happened to enrich the population in this mutation. So the implications of what we found were that we believe that the age of mutation supports the work from this group rather than the work that suggested that the mutation was roughly 50,000 years old. That really fits with the distribution of cystic fibrosis and the current population. This more recent appearance, if you will, really does help explain the high concentration of this gene and the prevalence of the disease in western Europe, particularly northwestern Europe, and knowing when the mutation appeared and getting a similar age in three widely separated populations gives us new insights. It sells us that this mutation was moved around, dispersed through Europe during the Bronze Age and there definitely were active migrations occurring then. We can also say, if this is when it appeared, that these people were exposed to a variety of different stresses. This is the Plague of Justinian. It's a Roman plague, the Bubonic plague, probably the precursor to black death. And we're starting to do some analyses of what might have happened along those lines. So that sort of gives you an idea of part one of our research related to the when. Now, part two, we wanted to have a direct confirmation of this. We wanted to actually find, demonstrate the delta F508 mutation in archaeological specimens. And so we learned that there's plenty of archaeological specimens available in Europe and you can get your hands on them and, in fact, they're very, very enthusiastic, the archaeologists in Europe, about this kind of research that might have some relevance to health today. It's completely the opposite here in the United States where you can hardly study ancient burials because of federal laws that protect the Native American population. And so archaeologists in this country and very, very frustrated so they like to work with material they get from outside the United States. So we found early on that teeth, particularly molars, were very good for ancient DNA fingerprinting. So that's how we happened to get teeth from a variety of places. And that femur samples were really very good for radiocarbon dating and trying to assess the diets by looking at stable isotopes and also measuring trace elements. And we're particularly interested in toxic metals like lead and arsenic. I'm not going to have a lot of time to go through that. This field of ancient DNA research is relatively new. Only been underway for about 25 years. It's got a really checkered history. Its reputation, ancient DNA research, is really very mixed because impossible results were reported when this field began to develop in the mid-1990s such as finding human DNA prehistoric animals, this is impossible, and also human DNA that had multiple haplotypes. That means it didn't come from one individual. So like if you extracted DNA from this femur and you found multiple haplotypes, different genetic identities from one individual, that's impossible. So what was happening? It was contamination with modern DNA that was the problem. So most of the projects that have attracted the attention have been very small scale with one or a few human specimens like celebrity studies. You might have heard about the DNA analysis that was done on the Romanov family of Russia just to prove where that family had been assassinated. So, I say celebrity studies. And incidentally, that's essentially what it was with Osama bin Laden. That's one individual whose genetic identity was confirmed by a match using the techniques that we use. So contamination with modern DNA is a big problem. The risks are present from burial to bench. Archaeologists are not very careful about how they handle bones, for example. And so many of these studies have been flawed but you have to actually, in large scale studies like we've done it's crucial to avoid contamination with modern DNA. And we found that analyzing teeth rather than bones is much better because the DNA of teeth is encased by the tooth. The DNA is in the blood that's in the roots of the teeth and no archaeologist has touched the roots of the teeth. See, in other words, we're interested in the blood which is the source of the DNA and it's within the tooth. And if you clean up the outside of the tooth, which you can do, then you can avoid this contamination. So there are articles like this, if you're going to do ancient DNA research, do it right or not at all because you can really ruin your reputation if you publish something that's absolutely flawed. So this is what we've been working on at this international program. I'll just move through these slides a little faster. I actually got interested in this in 1993. I was on a sabbatical, you can see I was a lot younger here. I was on a sabbatical at the London School of Hygiene and Tropical Medicine, which is just down the street from the British Museum. And we had already established our newborn screening program and I was really interested in this heterozygote selective advantage and so I designed this project way back then and spent a lot of time planning it. I got distracted for a while while I was dean of the medical school, and as soon as I could, though, I picked this up and started working on it with my team. I really was very fortunate to meet Barry Cunliffe, or Sir Cunliffe, early on because he thought this was really a hot topic that had much broader implications than just cystic fibrosis. And one of the archaeologists at the British Museum, JD Hill, felt the same way. British Museum turned out to a fabulous source of specimens. Museum of London was even better. I'll never forget it, when I went to the Museum of London and the archaeologist there, Bill White, took me into the basement and showed me 17,000 skeletons that had been dug up over the years in London going back to when the Romans settled London. So these are skeletons through the ages. And where do they get them? It's when they put in the underground stations. They're digging to put in tube stations and the law there is if they come across any archaeological material they have to call the Museum of London. The archaeologists come out, they take the time to excavate the site, and so that's why the Museum of London has 17,000 individuals, skeletons, that can be studied. It's an unbelievable opportunity. It's incredible. I was really fortunate to meet up with Maria Teschler-Nicola early and work at the Natural History Museum in Vienna where there are 40,000 skeletons available for study and they're very interested in collaborating with people like us in these kind of studies. It just was like a gold mine when I got there. And also to work with, what's the best CF cystic fibrosis molecular genetics lab in the world, the lab of Claude Ferec in Brest, France. And here's Cedric Lamarche, my principal collaborator. And they were quite interested in this because this question about why the European population is enriched in this delta F508 mutation has been around for many years and it's been a mystery. So anyway, we got these Iron Age specimens, teeth and femurs, from a number of sites. And, again, every time I met up with an archaeologist who heard about the project, very happy to share their archaeological material. That would never happen in the United States. You could spend up to a decade trying to get access to just one skeleton in the United States. So we used teeth for our ancient DNA fingerprinting. We established two ancient DNA labs. One in Brest and one here in Madison out at the Clinical Sciences Center. Focused on delta F508. We also developed methods where we could determine the sex of the skeleton by DNA markers, male or female. We thought this would be very important as well. And then we did a number of other studies that I already mentioned to you. It's absolutely incredible how well preserved these skeletons are. This femur, for example, from 100 AD from a burial outside of the original walls of London. It looks perfectly intact. And, in fact, that's the case. And, generally speaking, the teeth are the best part of the skeleton. Because these people tended to die between age 20 and 40. So they died young before they wore out their teeth, and they didn't eat processed sugar. They didn't eat foods containing processed sugar, and there was no hard candy around then so their teeth were actually in much better shape than ours are. To be very honest with you, I can't believe the great quality of the teeth in most of the skeletons we've studied. And this gives you an example. Now here's one, those teeth are better than mine. I'm sure of it. I hate to admit it but it's true. And you can pick and choose, too. And we did that and we developed a method for grading these teeth. So this is a grade A tooth. We just know that this tooth is likely to yield DNA and more than enough DNA for all the analyses that we want to do. This is a B tooth. It's not quite as good. You can see the roots are a little porous here. There's been some wearing down of the surface here. And here's a C tooth. It's really in bad shape. And we know that these C teeth are very unlikely to yield any DNA for analysis. And we know this in part because we've x-rayed these teeth, and we've sort of compared what the x-rays look like. So these are teeth that about 2500 to 4,000 years old that we've x-rayed. And so this is where we get the DNA. This is where the blood was on the roots of these teeth and in the pulp cavity of the teeth. And you might say that must not be very much DNA. You'd be amazed at how much you can get out of these teeth. So this is the way we went at the project. We select high quality teeth on the site and we minimize the handling. We clean them, we decontaminate them, we process them, and then we quantitate the DNA. We find out how much we have, and then we amplify segments of the DNA to look for the CFTR mutation with a number of methods that we developed processing these teeth, making a powder. We actually use a mineralogy mill that they use to grind up rocks to make a powder. Extract the DNA. We quantitate it with an amplification with the PCR reaction. And then we use forensic STR kits that Omega makes. Same thing that was used on Osama bin Laden and we determine what the identity is and we also look for the three base pair deletion of the delta F508. And so that's the way we do this. I'm not going to go into a lot of detail but that's our target right there. And so we can amplify the region around it with some DNA primers and then we can look for it. And we also use these STRs, the forensic DNA analysis. This stands for short tandem repeats, which are unique for individuals, and we can also determine if there's any modern DNA contamination. So we've only had this happen one time. We know what the STR pattern is for the handlers of these specimens, like myself and our technicians, and only one time did we find the STR pattern of a technician. So we've been very good about avoiding contamination. And we've replicated over and over and over again to make sure we confirm our own results. And these are just examples of replicating. And so here's analyzing modern DNA. We use this as control. So this is blood from me, and this is a DNA signal from my Y chromosome which tells you that I'm a male. And here's my X chromosome. There's my X chromosome. So you know I'm a male because here's the X, here's the Y. I do not have cystic fibrosis mutation. How do I know that? The delta F508 mutation. This is the peak right here. It's a 90 base pair peak. And it's a single peak. And so I don't have the delta F508 mutation. And these are just like what was looked at for Osama bin Laden. These are the STR patterns that demonstrate what my genetic identity is. And these are unique. All right, here's an example from a mother of one of my patients. And she has no Y chromosome DNA. So she's a female. And you can see she has two peaks here. She has the normal CFTR peak and she has the delta F508 peak. And how do we know that? This is a 90 base pair peak right here and that's an 87 base pair peak so it's got the three base pair deletion. So she is a CF heterozygote carrier, and this is her STR pattern and it's different than mine so we know what her genetic identity is. And like I say, this is exactly how it was done with the DNA from Osama bin Laden to look at the forensic DNA pattern with STRs. So this is when we discovered the delta F508 mutation in ancient DNA from a burial in 351 BC. How do we know it was 351 BC? We did radiocarbon dating of the femur from the individual whose teeth showed us the delta F508 signal. So here's three of them. And this is not the best but there are two peaks here from a burial. We did the analysis in January of 2007. This is a burial from the France Hausen cemetery along the Danube. There is another burial, another individual, number 203. You can see the two peaks. And here is another, here's a confirmation from this individual. And there's the STR pattern. This is before we started using multiple signals. And this is not quite as good. So this is when we discovered that without any doubt the delta F508 mutation was present in this population during the Iron Age, 351 BC. And we sequenced just to show that it indeed was a three base pair deletion. And we looked at a number of these populations and from that particular, and we looked at the concordance with gender, from that population, incidentally, this first group where we had no DNA recovered, those were all C teeth. And that's when we decided to quit looking at those kind of teeth. We weren't going to get enough DNA. But from these others here we selected 10 teeth. In nine out of the 10 we got adequate DNA for analysis. Here we selected 29 teeth. 23 gave us adequate DNA. Here, in specimens from the UK, 19 were selected, 17 had adequate DNA for analysis. And three of this group of 27 had the delta F508 mutation. So that tells you it was one out of 10 in that cemetery from that small population. And I won't go into details on how we did the radiocarbon dating, but it worked quite well for us. We expanded this project. This is the lab in Brest. This is the lab in Madison at the Clinical Sciences Center. We've expanded this to a number of different populations. Here we confirmed our results in two different labs. We took from the same individual. We analyzed a tooth showing the two peaks in France, and here we did the same thing in the lab here in Madison in April 2009. So when, and these are coded specimens, so when you confirm this way and you can replicate it, you have authentic results and you're not going to be criticized when you publish these kinds of results. And I forgot, this is the delta F508 mutation and this is from the Iron Age. Ancient teeth. And that shows you that this is a female. See the X chromosome DNA, no Y chromosome DNA. And this is the STR pattern. Let's see, what else? Okay, so I'm going to summarize now, and I'd be happy to answer questions. So we know from our direct studies that delta F508 mutation was definitely present in these Iron Age people of a Celtic culture that were traveling around Europe. This is central Europe near Vienna along the Danube River. There wasn't any Vienna in the Iron Age but people liked to settle around the Danube River because it was an international highway. This was how peopled traveled and traded. So if the mutation was there with a frequency about one out of 10 in that population, there's no question that the disease must have been there. And if was present in the Iron Age of Europe in this population along the Danube, we think it must have been present earlier, somewhere between the neolithic period, which is like 5,000 BC, and the Bronze Age. But our studies with modern DNA suggested that it was in the Bronze Age about 2,000 BC. And I won't have time to go into this but we've also found that these populations show great variations in their diet and also we have found incredibly high levels of arsenic and lead in the bones of these individuals. The Romans who lived in London literally poisoned themselves to death. The lead and arsenic levels in their bones are the highest we've ever seen. And we now believe, particularly since there's a relationship between arsenic and the CFTR gene and its function, we now believe that it was protection against arsenic or lead toxicity that may very well have been the explanation for the heterozygote selected advantage. So that's where our project is now and that's where it's going. We're going to start studying cells in vitro.

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57:40,32 And I really appreciate your attention, and thank you very much for the opportunity.

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