April 25, 2013 – The Genomics Landscape a Decade after the Human Genome Project More:
Claire Fraser:
Thank you so much, Eric. Good morning to everyone.
I am delighted to be here. And I think that
it's particularly important in a field that
has moved as quickly as genomics to take these
types of opportunities, the 10-year anniversary,
the completion of the human genome sequence,
the 60th anniversary, the discovery of the
double helix, to pause and look back upon
just how much has been accomplished. And so
it's delightful to be part of the group doing
that today.
What I'm going to talk about today is the
gut microbiota. It is an essential component
of what it means to be human. And the information
that we have about our microbial partners
that make up the microbiota and the all of
the genes that they encode, the microbiome,
represent another spectacular success that
has come out of the work that went into getting
the human genome sequence completed initially.
Just a few little facts about what we know
about the microbiota today, and those of you
who are familiar with the field have probably
heard the statistics ad nauseum. But they
really do, I think, help put all of this in
perspective. It's estimated that we carry
with us, as human beings, hundreds of trillions
of microorganisms that live in very close
association with us. We are essentially born
sterile, but immediately after birth, a very
complex process of colonization begins, and
within a couple of years, these bacterial,
these — well, these multi-microbial communities
mature and take on the characteristics that
they have for most of the rest of our lives.
It is estimated that we, as individuals, probably
carry 10 times the number of bacterial microbial
cells as human cells; obviously, that doesn't
take mass differences into account, but if
you think about this just in terms of cell
number, we are really only 10 percent human.
And if you do some quick calculations based
on estimates of bacterial genome size and
gene density compared to humans, the numbers
suggest that we carry along with us 100 times
more microbial genes than human genes. So
just by sheer numbers alone, I think we need
to pay more attention to our microbial partners.
And there is a concept that has emerged that
I think has really taken hold and is critically
important, and that is the concept of us as
a super-organism, representing a combination
of human DNA and the DNA encoded by our microbial
What Eric didn't mention in his introduction
today is that NIH has taken the lead in another
international project to begin to characterize
the microbes that live in association with
humans. This is the Human Microbiome Project;
it's finishing up five years. There have been
some spectacular successes. There were a number
of papers published last year, summarizing
the efforts of large numbers of laboratories,
many of them that were also involved in the
human genome project. And if you look at this
PCA plot here, this just looks at the differences
in the content of the microbial communities
that you find in different human environments.
And the environments are color-coded, and
you can see that we have co-evolved with our
microbial partners to the point where we find
now very distinct communities associated with
different areas of the human body. There are
a small number of bacterial phyla that you
see associated with humans. This is a very,
very high-level view, and when you get down
to lower taxonomic levels to look at genus
or sometimes species levels when you can,
we see that there is tremendous variation
among individuals. And I think at this point
we believe, at least in terms of the types
of organisms that are there that have come
out of our ability to do a molecular census,
I think there is good agreement that there
is far more variability between any two individuals
in terms of the microbes that we carry than
there is difference in our human DNA. And
the significance of that perhaps will become
obvious as I go on in my talk.
I'm going to now focus, for the rest of the
talk, on what happens in the gastrointestinal
tract. The GI tract is very heavily colonized.
It represents the most diverse bacterial community.
The estimates, depending upon the methods
used, suggest that we have many hundreds,
if not thousands, of different bacterial taxa
that live with us in our GI tract. There are
two major phyla: the Bacteroidetes and the
Firmicutes. And again, as I said before, we
see a lot of variation among individuals.
And in individuals, we can see variation overt
time. And that is thought to be due to a lot
of factors, including aging, diet, exposure
to antibiotics. And I'm going to talk a bit
about this more in the talk.
One of the important concepts that has come
out of this work is the idea of a core microbiome,
or a core microbiota. The question is, do
all of us as humans share key taxa that must
be present in order for these communities
to carry out the functions that are associated
with health. Although when you look at the
great variability, there's another way to
think about the core, and that may be a core
set of functions that can be carried out by
different suites of organisms. And I think
the jury is still out on that, but this has
emerged as an important concept.
Another concept that has come out of work
on the human gut microbiota, this was originally
reported by a group in Europe, the MetaHIT
Consortium studying the GI microbiota, and
has been now picked up on, and confirmed,
and refined by a number of other studies,
and that's the idea of enterotypes, that there
are distinct community types present in the
human population, present in the human GI
tract. Each of these different enterotypes
is — can be defined by a dominant genus,
and these are the three dominant genera here
that were initially described by the MetaHIT
study. If you don't know microbiology, it
doesn't really matter. The concept here is
that perhaps we could take everybody in this
room, do a characterization of your gut microbiota,
and place you into a limited number of community
types. What's been suggested is that, in part,
these different community types may be driven
by diet, although that isn't seen in all studies,
and it may likely be driven by human genotype.
And the factors driving the concept of enterotypes
is not known.
It's also, I should say, somewhat controversial.
There are some who believe that there are
not distinct groups, but it's more of a gradient.
But I think everyone would agree that if you
go in and look at anybody's gut microbiota
at any point in time, you will see that it
is characterized by, usually, one dominant
genus of bacteria.
We have coevolved with our gut microbiota
very much, we think in large part, because
of the important role that it plays in nutrition
and metabolism. The bacteria that live in
our guts have the capability to break down
indigestible carbohydrates, the plant polysaccharides
that we take in. This has important consequences,
although they may be obvious. One is that
it certainly increases the efficiency through
which we can acquire nutrients. The ability
to break down these compounds probably also
ensures that these microbial communities have
a constant source of energy themselves. And,
in turn, there have been a number of very
interesting studies that have begun to suggest
that diet and nutrition can shape the microbiota.
These communities are very dynamic. You see
differences in omnivores versus carnivores
versus vegetarians. If you look across the
animal kingdom, you can see differences just
by changing diet. And so these are just observations
at this point, and I think a big question
is what does all of this mean, and hopefully
we will continue to make some good progress
in figuring that out.
But what I want to focus on for the rest of
the talk is also the fact that the interactions
between us and our gut microbiota go beyond
metabolic functions. And very likely, a critical
role that these bacterial partners play is
in modulating and helping to shape the immune
system and vice versa. The gut is the largest
immune organ in the body. In humans, if you
think about this, there are probably — it's
probably a 10-micron layer of epithelial cells
that separates the lumen of the gut where
all of these organisms exist from the underlying
immune cells, the lymphoid-associated tissue
in the gut, and the surface area in the gut
is estimated to be about 200 square meters.
So that's a lot of area for interaction between
our immune system and these commensal organisms
that have to live in peace with us for the
rest of our lives.
There are a number of ways, if you look from
the inside out, that this happens. There is
a mucus layer — two-phase mucus layer that
provides a physical barrier. We also know
that antibacterial proteins can be secreted
by some of the cells lining the GI tract,
as well as the release of secretory IgA by
the immune system. So that helps to keep our
microbiota in check. And conversely, the microbiota
also impacts immune system development, and
a lot of these insights have come from studies
on germ-free mice. In the absence of any microbiota,
you don't see normal formation of the organized
lymphoid tissues that are associated with
the GI tract. There is altered regulation
of innate cells, and there are also changes
in systemic immune cells and systemic immune
responses. I don't have time in the presentation
today to go into this in a whole lot of detail,
but there is an incredibly well-balanced homeostasis
between our commensal microbes and the GI
immune system under normal healthy conditions.
So what I'm going to talk about today, then,
is how we respond — how the gut immune — how
the gut microbiota responds to the presence
of an enteric pathogen. In this case, we're
going to be looking at shigella; this is — which
causes the disease Shigellosis. This is a
mucosally-invasive bacterium that is associated
with food poisoning. Terrible symptoms: bloody
diarrhea, fever, stomach cramps. It is transmitted
through a fecal-oral route, and you see there
are a lot of deaths, particularly in children,
in developing countries from shigella and
other enteric pathogens.
shigella is a pathogen; in terms of its ability
to cause disease, is restricted only to humans
and non-human primates. And one of the most
important models for studying shigella in
terms of non-human primates is in the cynomolgus
monkey. This is an important animal model
that has served as a good system, and a lot
of vaccine development work, including attempts
at development of vaccines against shigella.
And so most of what I am going to talk about
now has to do with some studies in cynomolgus
monkeys. It is much easier to do intervention
studies, to do challenge with wild-type shigella
in the primate model as opposed to trying
to do these studies in humans.
And when we look at what's been done in the
efforts to develop an effective live-attenuated
vaccine against shigella, and a lot of this
work has been done by Mike Levine and colleagues
at the Center for Vaccine Development at the
University of Maryland, there has been some
slow progress made, but successful vaccine
development has been stymied by a continued
finding that regardless of the vaccines that
are tried, regardless of vaccine regimen,
there is tremendous variability in vaccine
response, particularly if you look across
global populations. And this is not unique
to shigella vaccines; this is a somewhat more
universal theme in vaccine development as
a whole. The causes for this variability are
not known, but we can begin to make some assumptions:
underlying genetic differences in the hosts,
diet, environmental differences. It's been
mentioned already today that many people in
developing countries carry a great parasite
burden in the intestine. This may, in part,
contribute. But one of the things that has
not really yet been examined is the potential
role of the gastrointestinal microbiota in
vaccine response. And this was the goal of
the study that I'm going to tell you about.
So this was — we piggybacked onto shigella
vaccine trials that were being carried out
in cynomolgus monkeys. These were looking
at efficacy of oral live-attenuated vaccines.
And I will get to the study design, but we
characterized the gut microbiota in stool
samples, and stool samples have been used
very often in these kinds of studies as a
surrogate for what is going on in the intestine.
And we can argue whether that's fully appropriate
or not, but it's clearly much easier to collect
stool samples than to go in and do biopsies,
particularly over repeated sampling. And so
we were interested in characterizing the gut
microbiota in these monkeys post-immunization
and post-challenge with wild-type shigella.
With the big question being, how does, or
does exposure to an enteric pathogen affect
the intestinal microbiota; and conversely,
does the composition of the intestinal microbiota
possibly contribute to the outcome following
immunization and challenge?
What we used to characterize the gut microbiota
was the 16S ribosomal RNA molecule. This allows
us very quickly and easily to take a molecular
census of all of the organisms that are present
in the gut microbiota. Most of these organisms
cannot successfully be grown in culture, so
we need to be able to bypass that cultivation
step, and we can go directly to the characterization
of these organisms through looking at DNA.
The 16S ribosomal RNA gene is quite useful.
This is a two-dimensional representation of
the structure, and these loops here that you
see that are highly conserved and are essential
to the function of the ribosomal RNA molecule
come about through the presence of highly-conserved
regions of the gene that are involved in base
pairing here, interspersed with highly-variable
regions. So we can design PCR primers against
the conserved regions of the 16S molecule,
amplify across the variable regions, and this
gives us incredibly useful information for
making taxonomic assignments and constructing
phylogenetic relationships.
So we looked at a number — we looked at monkeys
in a number of studies. I'll go through all
of these, but we're really only going to focus
on a couple of studies, then, for the rest
of the talk. In Study 1, this was nearly a
three-month study, where two different live-attenuated
vaccines were being evaluated for efficacy,
compared to animals that received PBS as a
control. In order to vaccinate these monkeys,
they are anesthetized, and the bolus of shigella
vaccine is administered through oral gavage.
So in the PBS-controlled monkeys, they were
getting PBS only as a vehicle; they were not
getting any attenuated shigella. There were
two immunizations here shown in yellow, followed
by a wild-type challenge at Day 56 in red.
Study 2 was a separate study with a different
set of monkeys. This was looking at one of
these two vaccines but a different vaccination
regimen: four doses given over the period
of a week, again, followed by wild-type challenge
at a month. Study 3 was looking at naïve
monkeys that didn't receive any immunizations.
They were just challenged with wild-type shigella.
And Study 4 was a group of control monkeys
in quarantine that received no handling, no
intervention whatsoever. But we are going
to be focusing mostly on Study 1 and Study
So we wanted to first characterize what organisms,
what types of organisms were present in the
GI tract of the cynomolgus monkeys. This is
the relative abundance here at the phylum
level. We see the Firmicutes and the Bacteroidetes
as the two major phyla, but they are present
in very different proportions from what you
see in humans. If anything, in humans, the
relative percentage is switched and more even.
If we look, then, at the genus level, these
are, again, looking at data we collected from
all of the monkeys over time, the most predominant
genus that we see is Lactobacillus, Streptococcus.
These are all in the Firmicutes. Prevotella
was the major member of the Bacteroidetes,
but the point here is that this community
composition was different than humans in many
If we then look at trying to ask what, perhaps,
represents a core in the monkeys, and we defined
a core, I believe, at 85 percent: any organism
that was present in 85 percent of the samples
at greater than 1 percent mean relative abundance.
These are — again, this is what we saw at
levels of prevalence, and these are monkeys
that are part of the different study groups
here, color-coded by the different study groups.
And we see that there were some differences,
and I'll get into those differences in a bit
more detail in a moment.
Just to go back, again, to the concept of
enterotypes in terms of the GI microbiota,
this is what we see in humans, three different
enterotypes. These are data from the MetaHIT
study I mentioned previously. We've done a
lot of work with the old order Amish in Lancaster
County, Pennsylvania in an obesity study looking
at the potential contribution of the gut microbiota.
We see essentially the same enterotypes in
the Amish. We and others have done other studies,
and depending upon the populations, we don't
necessarily see all three of these, but what
we do see tends to fall into one of these
three different groups.
So one of the first questions we wanted to
ask was whether or not there were enterotypes
within the cynomolgus monkey gut microbiota
that we could identify as a way to begin to
try and think about how to map the results
from this study onto results from human vaccine
trials. This is, again, looking at all of
our samples here. This is a three-dimensional
PCoA plot. We saw four different community
types here that we numbered one through four.
These are the characteristics of the enterotypes,
and, again, they are each characterized by
the presence of one or two more predominant
genera; very different from what we see in
humans, but this concept still holds, that
this is not necessarily a continuous gradient
of variability.
When we looked at the four community types
here with a measure of overall diversity — this
is the Shannon Diversity Index from — really
derived from the field of ecology — we found
that there was a very high-diversity community
type, Community Type II here; a low-diversity
community, Community Type III; and Community
Types I and IV were similar in diversity.
Community Type II, the very high-diversity
community, is characterized by a high level
of two bacterial taxa here: Prevotella and
an unknown taxa that's given the number 2159.
This contrast with Community Type III, where
this is a community type that is dominated
to a very great extent by a single genus,
Lactobacillus; therefore, because it's a single
genus that's present at a high level, the
overall diversity is low. So our analysis
revealed that these four community types,
each dominated — each characterized by a
dominant genera. So the concept still seems
to hold in the cynomolgus macaque model.
We wanted to ask what might be driving this
diversity in the monkeys, and certainly one
of the suggestions in humans is that diet
may play a role. We didn't think that that
necessarily would be quite so important in
these monkeys because they had come in from
different vendors but all were in quarantine
for 90 days before these studies began, and
then over the course of the studies, were
being housed under identical conditions, fed
the exact same diet, and same light/dark cycles.
So some of the potential variables that are
thought to contribute in humans didn't seem
like they would be playing such a big role
in this particular model.
So we immediately went to ask whether there
was genetic diversity in the cynomolgus macaques
that we might be able to identify. And this
was based on a lot of previous work, which
has indicated that there are, indeed, allelic
differences within cynomolgus macaques from
different geographic origin. And, in fact,
macaques from Indochina and Indonesia show
a very high level of diversity, particularly
within the MHC regions I and II, and these
are contrasted with macaques from Mauritius,
which are geographically isolated and have
been shown to have a restricted genetic diversity
in the MHC region. And one can, perhaps, think
about these different subpopulations of cynomolgus
macaques almost in the same way that we think
about outbred and inbred animals.
The MHC haplotype in macaques has been shown
to be very important in disease susceptibility,
particularly in studies with simian immunodeficiency
virus. And so we carried out both whole-genome
analysis using short tandem repeats, and genotyping
analysis, looking specifically at the MHC
region to try and get a sense of whether there
was genetic diversity in our monkey populations.
And this is what we found. This is a PCoA
plot here. This is looking at genotype analysis
using 24 non-MHC short tandem repeats. And
we found that, indeed, as had previously been
described, the Mauritius — the animals that
were from Mauritius — and this was information
that we both got from the vendor, and also
confirmed with these studies, clearly clustered
separately from the rest of the monkeys from
Indochina and the Philippines. So we knew
that we were working with monkeys of different
geographic and different genetic backgrounds.
So when we looked at the MHC alleles, we looked
at seven loci across the class I and class
II region. And we then used that data to construct
a phylogeny based on the MHC repertoire. And
again, we saw that the monkeys from Mauritius,
shown here in green, clearly cluster as a
group, distinct from the rest of the monkeys
that we were using. So we knew that we were
working with a mixed population of monkeys.
So now we went on to ask, given that information,
given that we see differences in the microbiota
that actually map — and what I didn't say
is the Community Type II, the very high-diversity
community type, maps to the monkeys from Mauritius.
So we now went to look at the effect of vaccination
on the microbiota in these different animals
to ask the question, "Does vaccination or
challenge with wild-type shigella alter the
gut microbiota in any way, and are the observed
changes the same for all populations?"
One of the first things that we noticed was
that in Study 1 — and these were all monkeys
from Indonesia and Indochina with greater
MHC allele diversity. This is the Shannon
diversity index; again, a measure of the diversity
of the community. It takes the number of different
organisms, the relative abundance into account.
This is the Shannon diversity pretreatment.
We found that after the first immunization,
the Shannon diversity decreased; decreased
yet again following the second treatment,
and then came back, following the challenge.
And this is comparing the data to what we
saw in Study 4 monkeys, which were our control
that didn't receive any handling.
What we found in Study 2, however, was very
different. This was — these were the animals
that received four immunizations. There was
no change in Shannon diversity whatsoever.
So we saw that in terms of the response of
the gut microbiota to immunization, to handling,
there was, indeed, a distinct difference between
the Study 1 and the Study 2 animals.
Now, this is going to be a very busy slide,
and I will try and go through this step by
step so it makes some sense. This is probably
one of the most important slides in terms
of all of the data here, and it's really a
compilation of all of the data that we had,
putting our microbiota data together with
immune data.
In our Study 1 monkey, this first set of bars
shows the community type in the monkeys, color
coded here, 1, 2, 3, 4. You can see — or
where we did — white being where we had no
sample. And what you see here, as I had just
described, that after the first immunization,
in some of the monkeys, we begin to see a
shift from Community Type I to Community Type
III. This is the low-diversity community dominated
by Lactobacillus. Following the second immunization,
essentially the communities in all of the
monkeys shift to this low-diversity community
type, shown by the light purple. When the
animals are challenged with wild-type shigella,
we see a shift, yet again, to Community Type
IV, shown in the dark purple. And this — I
will describe the characteristics of that
to you in another couple of slides.
So we are seeing a continued shift. And what
I should point out is that we see this regardless
of whether the animals are given the vaccine
or given PBS. And so we don't necessarily
think that this is an effect of the vaccine,
per se. We think that this is — what we're
probably seeing here is an effect of the stress
of the animals being anesthetized and having
to go through the intervention. We actually
had stored blood. We went back, and we were
able to measure cortisol levels, and found
that cortisol levels peaked in the monkeys
after each of these interventions. And stress
has been described previously to potentially
alter the composition of the gut microbiota.
Next, we look at clinical score. What happens
following immunization or challenge with wild-type
shigella? Green is normal. In terms of what's
looking at — being looked at here in the
clinical score, this is diarrhea, diarrhea
with blood. For the most part, nothing happens
until the animals are challenged with wild-type
shigella. And then in some of the monkeys,
we begin to see some clinical symptoms, as
shown by yellow, or, in a couple of cases
here, orange or red, which was really severe
clinical symptoms. And again, we see this
across all of the monkeys, and we see this
in association with the emergence of this
Community Type IV.
In Study 3, we see something similar. These
are the animals that were just challenged
with wild-type shigella, no immunization.
You immediately see the appearance of these
altered communities and clinical symptoms
in at least two of the animals.
And now if you compare this with Study 2 — these
are the animals from Mauritius that are clearly
different in terms of their genetic background.
The community type here is represented at
baseline by either I, or a large number of
the high-diversity Community Type II. Even
giving four immunizations over the course
of a week, there is essentially little, if
any, change in the gut community composition.
That also — nothing changes following challenge
with wild-type shigella, with the exception
of one sample here from one monkey.
Now we fold in the immunology data. And we
see that when the animals that are given the
immunizations, when we look at IGG or IGA
levels against LPS, we see an increase in
antibody levels. We don't see this in the
PBS monkeys, and we see a more robust response
following wild-type challenge. This is the
case as well in the Study 2 monkeys.
But what I think is most important here is
that in the Study 2 monkeys, it's not so much
the immune response. But in the study 2 monkeys,
particularly the monkeys that only received
PBS, they did not get sick following exposure
to wild-type shigella. And these are the monkeys
that harbored a very different high diversity
gut microbiota.
So I think we've summarized all of that. And
in the interest of time, I just want to keep
Community Type IV: We seen an increase in
very rare members of the community here that
seem to be associated with the clinical symptoms.
And one of the — so one of the, I think,
very important conclusions that we have here
is that only the Study 1 monkeys exhibited
clinical shigellosis. The immune response,
as we measure here — and we looked at antibodies
to other components — does not seem to be
a determinant of protection. So what is? And
we are proposing that there perhaps is a potential
role for the microbiota in mediating this
So we took — we did one more set of analyses.
This is to look to see whether there are any
correlations between the strength of the immune
response, the type of the response, and the
microbiota using the local similarity alignment,
which allows one to look for time-dependent
correlations. In Study 1 monkeys, this is
the network that emerged. It's very dense.
It's very complicated. It's probably, in part,
related to the changes that we saw in community
type. But we did see some correlations here,
positive correlations between antibody level,
stool scores, and particular members of the
gut microbiota.
This is what we see in Study 2. And in the
interest of time, I can't go through this
in a lot of detail. But what we did see were
some shared correlations among the studies,
and that a protective response was associated
with three members of the gut microbiota in
these monkeys. However, we realize that the
differences in vaccine regimen may make it
hard to figure out how all of these are related
with these data sets.
So, in conclusion, what we have seen are the
presence of different enterotypes in the monkeys.
Two of these seem to be associated with health.
Two of them seem to be transient, that are
associated with intervention or with challenge
with an enteric pathogen. But I think what's
most important here is that these different
enterotypes are present in macaques from different
geographic origins that are clearly different
genetic backgrounds.
Vaccination in challenge induced an immune
response in both studies. But the immune response
does not, in any way, explain the outcome.
And so we are now very much interested in
further exploring the potential role of genotype
in shaping the composition of the microbiota.
We saw that the high-diversity enterotype
was — seemed to be protective against shigella,
or was at least associated with a lack of
clinical symptoms.
And one other comment. In another unrelated
set of studies in humans, looking at response
to live oral-attenuated salmonella vaccination,
we see that a very high-diversity gut enterotype
in humans in these studies seems to be associated
with a more robust immune response.
And I think I would then just argue that based
on these data, particularly for studies like
this, it is probably critically important
to begin to think about folding in the microbiome
and information on the microbiome on future
studies. And I think this leaves open the
possibility that some of the differences that
have been well-described in efficacy from
vaccine trials in populations around the world
may be related to what's going on in our gut
and the role of our microbial partners.
And with that, I want thank my collaborators
on this study. And I — if there's any time,
I'm happy to take questions.
Male Speaker:
I'll ask — is anybody coming to a microphone?
So, Claire, technically, is the — at one
point in time, there was a lot of interest
in getting technologies for sequencing to
be able to get at single microbes as opposed
to populations. Is that still sort of a cutting-edge
need, or is the computational side of this
equation probably the more challenging immediate
Claire Fraser:
I think that's a great question. And I would
say that both of those represent very pressing
needs. The ability to do the informatics is
absolutely essential. But at some point, as
we want to begin to further dissect what's
going on, we need to be able to move away
from studying these communities as assemblages
of unknown [spelled phonetically] organisms
to begin to map function to specific cells
or specific types of cells. So —
Male Speaker:
And without the ability to culture them, you're
going to want to be to —
Claire Fraser:
You're still going to — yes.
Eric Green:
And one more question right there.
Male Speaker:
Thank you for that talk. Wonderful, as always.
I was just wondering if anyone is looking
at these enterotypes across phylogeny in the
primates to see if there are any central evolutionary
messages there.
Claire Fraser:
Those studies are just beginning, but I have
not seen anything published yet in any sort
of systematic way. Enterotypes have been looked
at in Rhesus macaques, and there's evidence
for enterotypes, but in the broad kind of
study that you're describing, I think that
is work still to be done.
Eric Green:
Okay. Well, thank you, Claire. We will —
We'll now move to the next talk, which is
not the one showing up on this monitor, but
rather Jeff Botkin is here from University
of Utah to talk about Whole Genome Sequencing
in Newborn Screening: What are we screening
for? Jeff.

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