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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
from 1984 through July 2006. of Wisconsin Medical School
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
Screening Project. Cystic Fibrosis Neonatal
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
to talk about cystic fibrosis. like to welcome Dr. Farrell here
Thank you. [APPLAUSE]
thank you very much. >> Phillip Farrell: Well,
I've been working on for several I really appreciate the opportunity to visit with you tonight and summarize a project
years. And I'll tell you why I got into
these bones here and teeth. I'll tell you what they are in it and also explain why I have
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
Laboratory of Hygiene on some very advanced methods of trace Shafer at the Wisconsin State
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
of cystic fibrosis. I'm going to explore this in to understand the ancient origin
archaeological genetic studies that are unique and associated
studies, such as radiocarbon
dating and trace element analysis, and ultimately what we
presumed heterozygote advantage for individuals who have one would like to understand is the
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
have at least one mutation in the CFTR gene. individuals that are CF heterozygote carriers as they
fibrosis, epidemiology, and newborn screening for CF, and I'll explain that to you in a because offew minutes. in cystic
So what are these bones and teeth?
This would be the knee joint Well, this is femur, as you can see.
And this bone is about 1900 years old. And there's the head of the femur.
which is a Roman city now known as London, and this is from a IRoman citizen that was buriedf the origiabout 100 AD.Londinium,
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
The distal end, the far end, is broken, and there's some knee joint is broken off.
femur. contamination with soil here. And there's the head of the
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
northeastern Italy to settle Vienna through the Alps and to
that area before it was conquered by the Romans. And here's a tooth from the
natural history museum in Vienna
extraction of ancient DNA and analysis by molecular genetics Iron Age Celtic burial again. And we use these teeth for
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.
collaborators. I want to acknowledge all my
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
chemist for trace elements. This Inserm unit is a research world's most expert analytical
unit in Brest, France. And all these individuals have
worked on it. Several people here at UW Madison.
Research Lab doing radiocarbon Tom Stafford from the Stafford
Cunliffe, or I should say Sir Barrington Cunliffe because he's Probably the world's foremost European archaeologist, Barry
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.
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
So these are some of our Ramsel at the University of Vienna.
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.
cystic fibrosis mutation in order for their child to have cystic fibrosis. So both parents have to have one
two cystic fibrosis mutations. You have the disease if you have
The incidence of the disease varies depending upon the ethnic
So among white Americans, one in or genetic background.
every 3,000 babies born in this country will have cystic fibrosis.
And Hispanic Americans about one
Okay, there we go. in 6,000. I'm having trouble keeping this mouse going.
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,
pancreas, which is a digestive inadequate function of the
organ, and that causes intestinal malabsorption and
Secondly, chronic lung disease, which is potentially fatal. And then, finally, the third malnutrition.
high concentration of salt in the sweat. principal manifestation is a
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
involved. It's difficult to understand, or lungs involved, and you would have the gastrointestinal system
involved and why there's other it was prior to 1989, why these three organ systems were
clinical manifestations, but a major breakthrough occurred in
the 8th of September, 1989, discovery of the principal 1989, published in Science on
mutation or the F508 del cystic fibrosis mutation, which is known as the delta F508
discovering the principal mutation. An70% of cystic fibrosise discovery of the gene by
It's an interesting mutation. It's a three base pair deletion at codon 508. cAnd 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
this protein does is simply facilitate the transport of chloride ions and, secondarily, sweat glasodium ions. and what
So at the 508th codon, if there's a three base pair deletion, you get a mutant
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
So the CFTR gene encodes a France, for example.
chloride channel protein and the abnormality is defective
these abnormalities in the lungs chloride transport which causes
and the pancreas, etc. So quite a number of organ
you'd think this would be a very systems can be involved, and
easy disease to diagnose, but if you look at the age of diagnosis
States, this is 21,588 of the roughly 30,000 CF patients in the United States, what you find among CF patients in the United
patients are diagnosed in the first year of life but about is that about two-thirds of the
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.
mention is that we know that 5% or 10% of children with cystic And the other thing I should
method of diagnosis. fibrosis can actually die undiagnosed by the traditional
And we also know that females, girls with CF are diagnosed at
And that's the reason why we got an older age than boys.
So we've worked on this with an NIH grant since 1984 year at the diagnosis through newborn screening.
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
And this newborn screening for street.
cystic fibrosis involves DNA analysis or molecular testing.
that. And the test we developed here I won't go into the details of
on the Madison campus is now used around the world so that in
the year 2010 about eight million babies were screened for
regions that are colored in green, I mean in red. cSo this is the United States,n many of the provinces of Canada.
I want to tell you why Ireland
is green here. I spent quite a bit of time in
from, because Ireland has the highest incidence in the world Ireland, where my ancestors are
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
underway I would color Ireland the UK system, but I promised them that if they got a program
green on this slide. [LAUGHTER]
to try to get 40 shades of green And I told them I was going
because of the song "40 Shades of Green" but decided to go with one shade of green.
Newborn screening actually began
very concerned about the poor icondition of children being, diagnosed in this state because hof the delay in diagnosis, and
we thought there would be a better way, namely early
That's what we got into. diagnosis through newborn screening.
positives. But when you do a newborn screening test or any kind of screening test you get false
Not every test that's positive means that the baby has cystic
And, of course, the same is true fibrosis.
for any kind of screening like screening mammography.
are false positives. About 7% of positive mammograms
That is, the woman does not have cancer but had something
It's about the same for PSA screening in men for prostate cancer. About 10% of the positive tests
are false positives.
And newborn screening is In other words, the PSA is high but the man does not have prostate cancer.
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.
so that you accept false And so you set the cut off point
positives in an effort to try to avoid false negatives.
screening, about 5% of the So with regard to newborn
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
there's about 10,000 false about it. In the case of cystic fibrosis,
positive tests per year in the United States and about 1,000 true positives, that is babies
diagnosed with CF.
Yes. >> If you did a repeat test would it still be a false positive?
I'm talking about tests that wound up being repeated.
positive, the second one was In other words, the initial was
positive, and then after a positive screening test a baby
test, a follow-up test. And what I'm saying is those needs to come for a diagnostic
follow-up tests would be negative.
So in the case of cystic
fibrosis, here's the typical thing.
baby comes in for the diagnostic We detect one mutation and the
test, which is the sweat test, and the sweat test is normal. So we know that baby is the CF
heterozygote carrier because the
by our DNA testing one mutation. baby does not have cystic fibrosis because the diagnostic test was normal, but we detected
So we know that that baby is a CF heterozygote carrier.
and my colleagues for a couple And parents have been asking me
of decades what does that mean for my baby?
that mean my baby is going to have a little bit of cystic fibrosis or have some kind of a My baby hasproblem?ation, does
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
copy of the sickle hemoglobin gene, are resistant to malaria. And what happened in Africa, carriers, that is they have one
Africa, is the severe and potentially fatal malaria there does not affect those that are particularly northwestern
And so over the centuries, that population has been enriched in individuals that have sickle carriers of sickle hemoglobin.
they're carriers of sickle cell disease. hemoglobin one mutation and
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
hemoglobin. You understand that? terminology a selective advantage for carriers of sickle
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
There's some selective advantage cystic fibrosis.
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
were great travelers migrating around 2500 BC. We think that people of the so-called Celtic culture that
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
genetic background. We've looked at some indirect ievidence, and we did some work is the ancestral background,
In fact, that's how I started out, studying transgenic mice that had one copy of the delta with animal models.
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 delta F508 mutation. the literature as to the age of
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.
Brittany have developed a method And then my colleagues in
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
process for this mutation. idea or at least develop some hypotheses about why. Why there was a selection
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,
correct, other infectious tdiseases, syphilis, influenza, some evidence but it's very limited and I don't think it's
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
there's this selective advantage could, in theory, explain why
for the CF heterozygote carrier. When you try to organize a
of take advantage of what we project like this you can sort
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
which we're now moving towards a molecular or cellular strategy
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
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.
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
frequent here and as you get mutation is most prevalent and
percent with delta F508, looking farther and farther from there, you see a decrease among cystic fibrosis patients and the
at these modern delta F508 percentages.
also sort of thought about how So to get at this where, we've
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
and testing out different possibilities like tuberculosis or metal toxicity. there's a selective advantage
this genotype persists, why the carriers persist. So, this comes from National And then we're interested in how
most of you know, we're all Africans originally. Geographic, and as you, I'm sure
Modern humans, Homo sapiens, developed, originally,
in Africa.
about 200,000 years ago. And we know that that was probably for modern humans, Homo sapiens,
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
into Yemen, present Yemen. Red Sea where it's narrow and they crossed over the Red Sea
And they were then living in the Arabian peninsula so you could
Americans and we're also Arabian say all of us are really African
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.
around 15,000 years ago. And they also migrated across the Bering Straits into North America somewhere probably
And so why am I bringing this up and why is this important?
until, by modern humans, until Well, if Europe was not settled
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
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
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
And we also obtained blood from their mother and from their father, and we showed that the mutation.
delta F508 carriers. mothers and the fathers were
And we did this from three regions of Europe with
cystic fibrosis. relatively high incidence of
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
And I won't go into the details on the methods but we looked at regions of DNA around the CFTR in Brest.
their haplotypes were, that is gene and we determined what
what's their ancestral genetic background, and then using a
developed in Paris called the ESTIAGE program for estimating very interesting program
age, we determined, we estimated, how many generations
back these individuals had a
individuals that I told you about, and we look at what their So you see what I mean? We've got DNA from all these
by this computer program to when genetic variation is, haplotypes, and then we go back
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.
Ireland, also very similar. So all of these are Bronze Age. That is, they're somewhere Brittany, almost identical.
And that's why we think that around 1500 to 2,000 BC.
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
during that period of time and example, off the coast? You can also think about what kind of stresses did they have
What else were they exposed to? It gives you an ability to sort of think through what might have diseases were around?
population in this mutation. happened to enrich the
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
widely separated populations getting a similar age in three
gives us new insights. It sells us that this mutation
through Europe during the Bronze was moved around, dispersed
We can also say, if this is when Age and there definitely were active migrations occurring then.
it appeared, that these people were exposed to a variety of
This is the Plague of Justinian. different stresses.
It's a Roman plague, the Bubonic plague, probably the precursor
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
We wanted to actually find, demonstrate the delta F508 a direct confirmation of this.
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
outside the United States. frustrated so they like to work with material they get from
So we found early on that teeth, particularly molars, were very
fingerprinting. So that's how we happened to get good for ancient DNA
really very good for radiocarbon teeth from a variety of places. And that femur samples were
dating and trying to assess the diets by looking at stable isotopes and also measuring
trace elements. And we're particularly
I'm not going to have a lot of interested in toxic metals like lead and arsenic.
time to go through that. This field of ancient DNA research is relatively new.
Only been underway for about 25 years.
Its reputation, ancient DNA research, is really very mixed It's got a really checkered history.
to develop in the mid-1990s such because impossible results were reported when this field began
as finding human DNA prehistoric animals, this is impossible, and
haplotypes. That means it didn't come from also human DNA that had multiple
from this femur and you found multiple haplotypes, different one individual. So like if you extracted DNA
So what was happening? genetic identities from one individual, that's impossible.
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
Osama bin Laden. essentially what it was with
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.
And so many of these studies Archaeologists are not very careful about how they handle bones, for example.
have been flawed but you have to actually, in large scale studies
avoid contamination with modern DNA. like we've done it's crucial to
better because the DNA of teeth is encased by the tooth. And we found that analyzing teeth rather than bones is much
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
the source of the DNA and it's interested in the blood which is
then you can avoid this wicontamination.. And if you clean up the outside of the tooth, which you can do,
So there are articles like this, if you're going to do ancient
DNA research, do it right or not
absolutely flawed. at all because you can really ruin your reputation if you publish something that's
So this is what we've been working on at this international
I'll just move through these program.
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
project way back then and spent a lot of time planning it. this heterozygote selective advantage and so I designed this
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
I really was very fortunate to started working on it with my team.
topic that had much broader implications than just cystic Cunliffe, fibrosis.because he And one of the archaeologists at
the British Museum, JD Hill,
felt the same way. British Museum turned out to a
Museum of London was even better. I'll never forget it, when I fabulous source of specimens.
the archaeologist there, Bill White, took me into the basement went to the Museum of London and
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
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
London. The archaeologists come out, have to call the Museum of
they take the time to excavate the site, and so that's why the
Museum of London has 17,000 individuals, skeletons, that can
opportunity. be studied. It's an unbelievable
It's incredible. I was really fortunate to meet
early and work at the Natural History Museum in Vienna where up with Maria Teschler-Nicola
there are 40,000 skeletons available for study and they're
very interested in collaborating
with people like us in these kind of studies.
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,
about the project, very happy to from a number of sites. And, again, every time I met up with an archaeologist who heard
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
So we used teeth for our ancient skeleton in the United States.
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,
We thought this would be very important as well. male or female.
mentioned to you. It's absolutely incredible how And then we did a number of other studies that I already
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
Because these people tended to skeleton.
die between age 20 and 40. So they died young before they
didn't eat processed sugar. They didn't eat foods containing wore out their teeth, and they
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
teeth. a method for grading these
than enough DNA for all the analyses that we want to do. We just know that this tooth is likely to yield DNA and more
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
And here's a C tooth. It's really in bad shape. And we know that these C teeth of the surface here.
DNA for analysis. And we know this in part because are very unlikely to yield any
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
And so this is where we get the DNA. we've x-rayed.
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.
the site and we minimize the We select high quality teeth on
we quantitate the DNA. handling. We clean them, we decontaminate them, we process them, and then
mutation with a number of Wemethods that we developede, processing these teeth, making a the DNA to look for the CFTR
We actually use a mineralogy powder.
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
I'm not going to go into a lot deletion of the delta F508. And so that's the way we do this.
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.
This stands for short tandem And we also use these STRs, the forensic DNA analysis.
repeats, which are unique for individuals, and we can also determine if there's any modern
DNA contamination.
for the handlers of these specimens, like myself and our one time. We know what the STR pattern is
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
There's my X chromosome. chromosome which tells you that I'm a male. And here'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.
It's a 90 base pair peak. And it's a single peak. And so I don't have the delta This isF508 mutation.t here.
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
So she is a CF heterozygote carrier, and this is her STR pair peak so it's got the three base pair deletion.
identity is. pattern and it's different than mine so we know what her genetic
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
femur from the individual whose teeth showed us the delta F508 How do we know it was 351 BC? We did radiocarbon dating of the
And this is not the best but signal. So here's three of them.
of 2007. there are two peaks here from a burial. We did the analysis in January
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.
F508 mutation was present in So this is when we discovered that without any doubt the delta
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
We weren't going to get enough DNA.
selected 10 teeth. But from these others here we
In nine out of the 10 we got adequate DNA for analysis.
23 gave us adequate DNA. Here we selected 29 teeth.
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
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.
See the X chromosome DNA, no Y And that shows you that this is a female.
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.
studies that delta F508 mutation So we know from our direct
around Europe. was definitely present in these Iron Age people of a Celtic culture that were traveling
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
of 10 in that population, there's no question that the with a frequency about one out
Age of Europe in this population disease must have been there. And if was present in the Iron
period, which is like 5,000 BC, alongand the Bronze Age.nk it But our studies with modern DNA sosuggested that it was in thec
Bronze Age about 2,000 BC.
And I won't have time to go into this but we've also found that
variations in their diet and these populations show great
individuals. also we have found incredibly high levels of arsenic and lead in the bones of these
The Romans who lived in London literally poisoned themselves to
The lead and arsenic levels in death.
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
So that's where our project is selected advantage.
now and that's where it's going. We're going to start studying cells in vitro.
And I really appreciate your
attention, and thank you very much for the opportunity. [APPLAUSE]