Welcome to the
Proteomics in Proximity podcast, where
your co-hosts, Dale Yuzuki, Cindy
Lawley, and Sarantis Chlamydus from Olink
Proteomics talk about the intersection of
proteomics with genomics for drug target
discovery, the application of
proteomics to reveal disease
biomarkers, and current trends in using
proteomics to unlock
biological mechanisms. Here we have
your hosts, Dale, Cindy, and
Sarantis. Welcome to another
episode of Proteomics in Proximity.
I'm your host, Dale Yusuki, with my
co-hosts, Cindy and
Sarantis. And this morning - Hey
there - and, Sarantis, what are we going to
be talking about today? Today, it's a
great paper, actually. There's a great paper
where it describes
ways of using biomarkers - protein
biomarkers - in early discovery of
inflammatory bowel disease (IBD),
and it is a great example of a crosstalk
between gut microbioma and
immunohomeostasis. And
it's really great because it combines a
multiomics approach where they have
investigated genetics
and proteomics, and
metabolomics, and also
they have done sequencing
of microbiota in order to
identify this crosstalk.
Cindy, what do
we know about the DUOX2? What is the
importance of this protein?
So the name, DUOX, stands for
Double Oxygen.
So it's a
marker within the genome. And,
in fact, this paper highlights
some variants in the
DUOX2 gene
that code for something that will
produce hydrogen peroxide,
H2O2, in the epithelial
lining. So in what is
the apical layer, or
the layer that's right next to
where essentially the results of
our food go by. And so
that production has an effect on the
microbiome. So this
idea that we might have a proteomic
biomarker that
is indicating something about
the host microbiome
interaction, that gut bacteria
that we all are so curious about and
has been the topic of so
many interesting
publications that we might have a protein
biomarker that's indicating something
about the dysbiosis or the
shift in that microbiome
that precedes inflammatory
bowel disease, is super exciting.
So that's what I see here.
Inflammatory bowel disease is
devastating. I mean, I personally know
three individuals who are affected. You
may know others, right?
Their digestion, their
immune system, and
it's really, really difficult because
you talk about the interplay
between the person,
their gut
microbiota, which is off.
It is different than regular people and
it's a complex organ, right? The
gut microbiome is essentially, I
mean, a lot of people talk about it as an
organ. This
group, including a group out
of Institute for Systems Biology,
does a fair amount of work on gut
microbiome, particularly
in their wellness
cohort. So, Cindy, why don't you tell
us more about this wellness cohort? I
understand this is Arivale.
That's right. So Arivale,
was a
company that spun
out of the Institute for Systems Biology. It
was based in Seattle.
And the
team focused on
collecting data
longitudinally
for a bunch of people
that just opted in for
reconsenting, so opted in
for updating their consent
and recontact. And
the great thing about that is they're coming
in regularly. They're
"well" when they start. And we'll
be talking about some other papers
that are coming out of this
exciting group.
But it offers an opportunity
to look retrospectively at
samples where individuals
who were healthy
actually will develop
some diseases.
As we age, we tend to
do that, unfortunately.
So it's
a powerful demonstration
of a wellness cohort
and having longitudinal sampling.
Like I
said, we'll double-click on that in this
podcast I think quite a bit. This paper
talks about over 2800
individuals from this cohort, but it was
larger. Was it almost 5000, something
like that? Yeah, that's right.
And of course, over time, you're going to
have people dropping out
inevitably. And so this is a really
great sample number. And the longitudinal
data that they have collected for
this group of individuals is stunning. So
they already had their whole genome
sequence. And then what? Did they
also sample their microbiome,
like from feces? Yeah,
exactly. But then they don't
need to necessarily collect data
on all the samples in all the modalities.
What they've done is they've
seen interesting things evolve in
this cohort and
then do specific,
work within
a subset. And that's
exactly what happened here.
So a subset of the individuals
from this
2800
subset of the Arivale
cohort had these
DUOX2 variants
and those DUOX2
variants had some,
I would
say this sort of
brings me back to this
idea that if we had -
what was the total number, Dale?
You pulled it up for me right before the
podcast. 300 and some
odd individuals? It was like 357
individuals.
With rare variants.
12.4%. Yeah, if we were going
to do a GWAS on
357 individuals
and compare that to control
samples, that's not a very
powerful GWAS by our
standards, in the genetic space. And
so making the association between
these variants and
inflammatory bowel disease, or Crohn's
disease, or this IBD
phenotype, might have been very
challenging. And so this is a novel
discovery, this DUOX2
association with IBD. And it
was made through an association that
includes
phenotypic data that is closer
to the disease, which includes
proteomics.
That, as well as the
microbiome. So the ability to
have this multiomics as
Sarantis highlighted and
amplify the power to detect
the relationship between genetics and
disease is something I think is
exciting. We end up with whole genome
data, right? And we also
have then all the clinical
laboratory measurements. You
have metabolites, where apparently they
measured some over
950
metabolites from these patients or
these volunteers, healthy
individuals, it looks like they used
three Olink Target 96 panels
for a total of 266
proteins. And then the microbiome, which
is a really important part, they're doing
16S ribosomal RNA
sequencing, which gives you this idea of
what the constellation of
bacterial species looks like
just from sampling
rsRNA. But then they do a
PheWAS. And maybe
we can walk through that a little bit?
What is a PheWAS
again? I don't know. Sarantis, you want to
talk about it, or cindy? I guess I'm
not so familiar with a PheWAS,
actually, to be honest.
I mean, it's one of the few
times I have seen that or come across that.
But I checked a little bit in the literature.
It has to do with phenotypic associations
and how these are correlating with the
changes that happens in the genome, for
example. And that's how
phenotype is like the correlation.
And the phenotype, their phenotype is
IBD. So they
have individuals with IBD from
these 2800 people, and they're
saying: What's associating
out of all this
multiomic data? You just say,
"Well, Dale, there's how many different kinds
of microbes in the stool?" We're
looking at 266 proteins and
950 metabolites and all
these other clinical measurements.
But then they zero in on this
one particular enzyme.
Or is it an enzyme?
This DUOX2?
Yeah, it's an enzyme. Enzyme.
And what is it doing?
What is the function of
DUOX2? Producing hydrogen peroxide
actually
kills bacteria.
And that is
adjusting the
microbiome, it
appears, right? I like to use the
language that we're
unveiling here in this paper -
these authors are unveiling - a little bit
about the mechanism. Super exciting.
But there could also be complexity
that they're yet to uncover
here.
As far as that particular
function, we have this idea
of hydrogen
peroxide, and we normally use hydrogen
peroxide to clean
up. It's antibacterial.
It oxidizes.
Exactly. What is
it then? DUOX2 is
normally doing what in the
body? I mean, DUOX2 in
the context of these
individuals is keeping a certain
type of gram-negative
bacteria away. We're talking
about homeostasis at the end. Whatever happens
in biology is homeostasis.
It's like the equilibrium. And
when you have the shifting of
equilibrium, then you have all of these
pathologies that's at the end,
according to Greek philosophy also, I
thought, let's talk about the equilibrium.
And keeping the equilibrium at the
end. That's pretty much what we have in our
body.
Looking there, yeah,
that's the thing. And looking a little bit
at the rare mutations, they have mutations of
frameships. Let's
say mis-sense. We have
different types of rare mutations in
the DUOX2 gene that
leads to loss of function.
They have
heterozygous individuals
with loss-of-function allele.
And, of course, this affects the level
of DUOX2. And, as a consequence, they have
seen this increase of IL-17C.
But just to
repeat what Sarantis said in terms of
homeostasis in the normal, healthy
gut with a normal,
functioning DUOX2
sort of enzyme right at the surface
of the gut lining, you've got
gram-negative bacteria held at
bay because of the hydrogen
peroxide. Apparently there's some kind of
segmented filamentous bacteria that's
also a healthy bacteria
that's associated
in that side of the
gut that's facing
the external environment.
But we have these individuals with these
DUOX2 mutations, where DUOX2
is not doing its job
correctly. This
PheWAS, this genetic
association. And then we have
the gram-negative bacteria
disrupting, right?
No more segmented
filamentous bacteria, gram-negative
bacteria invade. And then we have
this IL-17C being activated.
And so now we're talking
about no more
homeostasis.
It's dysbiosis.
Dysbiosis.
IBD,
right? The Greek
word of the day. There you go.
And then we have the same
cells that are being affected by
these gram-negative bacteria
producing IL-17C
in the
system, in the body that can be
picked up.
In mice, this
DUOX2 deficiency and
this IL-17 elevation
are associated with this
expansion of proteobacteria,
sorry, pathogenic
bacteria.
That was pretty
fascinating, right? Where they're able to
take the same system and draw these
conclusions even down to
invertebrates. I'm like, wait,
we're talking about DUO function in
invertebrates. Primordial,
they say, right? So you've got
your primordial
DUOX2 producing hydrogen peroxide.
You've got, in mice, this DUOX2
deficiency increasing IL-17C levels in the
intestine and
proteobacteria
elevation. And then you've got
in IBD patients this evidence. I
love
this approach to see
multiple lines of evidence in different
systems, especially in such a
historic pathway.
Historic meaning going back to
invertebrates in evolution. Evolution,
exactly. In C. elegans,
in worms. And here, even this whole
idea of, we've got this mechanism
that they're able to look at via - what
did they use - knockout mice? Yeah. And so
these mice were deficient in
DUOX2. They can go ahead and do a lot of
experimentation on those
tissues. And then there is something called
a colonoid. And I'm like,
colonoid? What's a colonoid?
Sarantis, what is
a colonoid? Again, I'm taking
the difficult questions now.
I try to be creative also in
the way that I'm answering.
Organoids. A lot of
buzzwords, different things. They called
organoids, I'm guessing,
that what they mean here
is just a culture. They take a
tissue and they culture this tissue, like
primary cell culture, in a way, but
keeping the integrity of the tissue. And
they can do measurements of the proteome
based on this tissue, I'm guessing this is
pretty much what they are doing there. I
see. And this is what? Colonoid. But
mini colons
from primary tissue. Mini tissues in
vitro or something, or 3D
tissue culture or something.
To take then
that finding in mice and then
being able to translate it
to humans, to say
that they believe that these
sort of deficient individuals.
And I thought one of the interesting figures
was just how rare these variants
were, but yet it
was in
certain populations it was
like the odds ratio was
really high. Was
it Ashkenazi Jews
as a population? Yeah, that's right. And
then there was another
cohort that it seemed
like these odds ratio
estimates were pretty high
because they were enriched. Right. These
populations have a lot of these
DUOX2 mutations in the
population. I just thought that was really
interesting, where you can have then maybe a
genetic test where
somebody's susceptible.
I will say, though, the
IL-17C levels
were high in
other subjects that didn't have that
DUOX2 variant enrichment. Didn't have
that DUOX2. So you have the
option, a possibility
suggested here, that you could do a genetic
test to see about those rare
variants. But in fact, the
IL-17C
levels were
elevated. There was a common
response, sort of a cascade,
that suggested this.
I don't know if they use the word
inflammatory, but in my mind that's what I
picture. And I think
this is
where I think the excitement around
proteomics is, but also with the
DUOX2, just going back to the DUOX2
variants, this is kind of how
I think about proteomics in the context
of genomics. If you
have a DUOX2
rare variant that suggests
an increased IL-17C
level - Dale, we'll just pick
you -
and I don't you see how
I end up in the good category? And
then you have an exposure
to a protein level over
your lifetime,
perhaps that I don't,
that exposure internally
in our body is a way that I sometimes think
about the relevance of
how we make these connections between
genetics and protein levels.
But anyway, that's just a
way of thinking about it that I think.
No, that was great. And then
I will add also the macrobiome factor
here because they have done a really crazy
experiment and they really like it in mice.
They use antibiotics to
eliminate actually the negative, the gram-
negative and they see that
IL-17C levels, they drop
down again. That means there's
really close talk with
microbiome.
One of the
applications of this paper is: could
fecal microbiota
transplantation, FMT,
or antibiotic treatment
for people
with this dysbiosis
where the gram-negative bacteria
are where they shouldn't
be is pretty,
how do I say, remarkable.
To think this is
a potential sort of mechanism,
by which,
again, knowing the mechanism
suggests therapeutics,
and even though the complexity
of DUOX2, and then the IL-17C,
and then there's a whole
bacterial story,
and then the immune system ...
There's T-helper cells.
CCR6, the chemo
attractant for lymphocytes,
there's FGF23
There's
just a cascading
mucosal
immunity, intestinal mucosal immunity
that has a protein signature
that I think can be
shifted and has that potential to
provide value. Well,
we should probably highlight the Crohn's and
Colitis Foundation, actually, in this
discussion. Right. So Crohn's and Colitis
Foundation, of course, has developed
a subset of
proteins that are
useful in helping
identify in a pediatric
cohort which of
those kids is
likely to develop complications
from their diagnosis of
IBD. So, again,
a longitudinal
cohort that they followed, and they came up
with this signature that helps them
have the potential to
score and insert a pause
between taking out a kid's belly, which
seems like a pretty good
use of a proteomic signature. So,
that's an important
story. We've got several
webinars from the team there.
Yes.
One of the interesting take homes, getting
back to the IL-17C
story ...
I'm reading from,
right before the discussion, high
IL-17C in carriers of DUOX2
loss-of-function variants is
not only a potential biomarker
for disturbed gut
microbe immune homeostasis,
but appears to reflect an early stage of
IBD
pathogenogenesis.
So here now we're talking about
a biomarker that could
be an early
predictive marker of
disease. And you think,
wow, by
studying genetics, by
studying phenotype,
by looking at multiomics
here it is: we come up with a
plasma
biomarker.
And you're doing
it simultaneously, or they're doing it
simultaneously. So I don't know if
you remember the Alnylum story, where
hereditary
amyloidosis has
this diagnosis
that's based upon a Gait
test, like your walking test in your
doctor's office.
So that is sort of a
difficult thing to diagnose,
but you can have the genetic test very
and know you've got a predisposition for it.
You just don't know if it will ever
penetrate and if you'll ever actually
be diagnosed with it. It
took time for them to identify a protein
biomarker that had promise for that
diagnosis. Here they're doing both at the
same time. So here you've
got a potential for a genetic test,
but maybe these people never
develop IBD.
And so you can do the
genetic test, and then you know who you will
have to monitor over time and again. This is
all research use only, right? But
we're talking about the potential for the
future.
What would be the clinical
utility of something like this? And
that
seems like -- A comment on that. Probably
more to a philosophical point-of-view, it's
like, at the end,
having a genetic test will
help for a prediction because there are so
many rare, let's say, mutations,
that at the end, you will never know the
real levels of your protein.
Because at the end, what people care about is the
proper levels of DUOX2, independent
of the mutation or not.
That means probably you will
need the protein biomarker,
the IL-17C, and all the
cascades that follows to be more
sure and more concrete in what we are seeing.
That's the way that they see, because so
many rare mutations, difficult to predict
the levels of the protein. I think that's
really important to have a plasma
biomarker to follow at this point.
Yeah. And
everybody's not going to use our 3K,
our Explore panel,
right? We're talking about something that's
developed for the clinic
that's very
specific. That would need to go through
regulatory approval to
get into clinical utility. But
yeah, very exciting.
And you think this could be
... well, in the
commentary, the researchers
saying patients and physicians
and scientists are looking
for how to unlock
this microbiome host
and then immune system
as like a Holy Grail
within IBD. And I thought that was
so interesting, that here it
is. They're looking at so
many different variables. When you
think about whole genome data and all the
genetics that could be evolved, and then you
think about all the different other
measures that they did, and then to
settle on a model,
a gene, a
particular plasma-based
biomarker, and then looking at how they all
interact, it's just
really interesting. And
down to the
types of microbiota
that are being
affected. It's
so cool. I mean, it's a
big data story, right? It's
how big data and a cohort that's
collected and consented longitudinally - I
know I've already said this - can drive
mechanistic discovery
to help define
disease biomarkers. And I think having a
biomarker is great, but having it where you
actually kind of have a sense of the,
like these multiple lines of evidence
- a mouse model -
having multiple
lines of evidence and some mechanistic
understanding of it, makes
it so much ...
I'm much more comforted in
seeing it implement in the clinic. And I
would hope it has the potential to move to
the clinic a little more quickly when we
actually have that mechanistic insight.
Sorry, go ahead. I was going to say that
after reading a paper like this, because it
was pretty dense, it's pretty intense,
right? It was,
I don't know, maybe 12 or 14
pages of heavy duty reading
and lots and lots of immunology,
which can be difficult to grasp. We had
mouse models in terms of dual knockouts. We
have all these
multiomics, and yet it's almost as
if, man, this is like the final word,
right? This is one
conclusive particular
avenue. And then it makes me think, wow, it
was only 250
or so proteins in the
plasma [that were measured]. What if they did an
Explore 3K on it, right?
And then I think, wow, I don't
know. Cindy, maybe you can shed light on
this. Does the UK Biobank collect,
the microbiome
samples from individuals?
Not to my knowledge. I haven't
seen any studies on it, which
I would expect. I know they're doing
metabolomics on
samples in the UK Biobank, but my
understanding is it's on plasma. Don't quote
me on that. But I just haven't seen
any data come out on the
microbiome on those patients. Because that
data set, that 16S
ribosomal data is super
important, because that's one-third of the
whole story here in terms
of how that microbiome
is interacting with the
host. Any
final comments on this? I mean, this is
such a cool paper, right? Even though it
was published in
2021.
This association
with inflammatory bowel disease and
its practical application, and a
wonderful multiomic story.
I mean, it'll be really
interesting to follow.
Yeah, it's a great story. And
so I'm thinking in the show notes, we
can maybe even put some of these proteins in
our Insight app and provide a link
to the ability to browse
through those, because I think looking at
these pathways might be really interesting.
And just to remind folks around our Insight
app, we've had a podcast
about this before, but essentially
it's a browser where you can very
quickly what I love about it is
that you can actually convert gene names to
UniProt IDs. And with
UniProt IDs, there's
only one UniProt ID per protein, whereas
we've got multiple gene names for
them. So that's really handy. But
also being able to just look at the
pathways and see which
proteins we have in our
panels, that you can get at that
pathway through multiple proteins versus the
ones that we don't have in our panels. And
that also helps people to identify the ones
they want us to put in the panels. And we
have a mechanism by which you can request
proteins to be included in our future
product development efforts. And
so we've talked
about how we're trying to cover the
proteome. We've got 3000 [proteins that our platforms measure]
today, but certainly our R&D team
is working hard on
covering more. Yeah, thank you for bringing
that up, Cindy.
Insight.olink.com is how to access this free
resource. There's some really great
tools inside it. Even if people wanted
to browse publications by
biomarker, right?
Very good point. Wouldn't that be
interesting to punch in
IL17-C and see what other
publications come up?
Which, by the way, is a low,
abundant protein, right? So if you look in
our validation data, also available on our
website that you can freely download, you'll
see that IL-17C is
in the dilution category of one-to-
one. So it's neat, right?
You're not
adding any dilution factor to it
to
manage how much
reagent might be needed to count
when our NGS readout or our
qPCR readout is used. And so
that one-to-one ratio suggests that it's in
that area that we've talked about before,
where Olink has really shined a light that
makes it much easier to see these low,
abundant proteins
than some traditional methods that we, of
course, have already made lots of great
discoveries using mass spectrometry, for
example. But this low abundant
area, I think the
number of publications we have in our
1100-plus publication
database, many of those are
focused on those one-to-one
neat proteins that are in that
low, abundant range. Speaking of show notes,
I'll be sure to include Dr.
Uh, Hurtada-Lorenzo's
talk from the Crohn's and Colitis
Foundation. This is where
they were looking at
pediatric Crohn's and colitis.
And it's a remarkable story in
terms of was it maybe 70
or 80 real-time
PCR markers that they had
developed from biopsy tissue, but
then they went to plasma and they found a
much smaller signature that's much more
practical. Instead of using
biopsy tissue and real-time PCR, here
it is. They're able to look at circulating
biomarkers. Some really exciting work.
And that team
has created, of course, it's a nonprofit
Crohn's and Colitis Foundation, but they've
created a ventures arm for that
entity in order to bring investment in
to take things like this to the clinic.
And they're funding the majority of
Crohn's and Colitis research in the world.
In fact, I was at a meeting in South
Africa, in Cape Town a couple of weeks ago,
and there was a poster on
IBD, and I talked to the
authors, and in fact, they were also
funded by the Crohn's and Colitis Foundation.
So it's just a
small world in some of these disease
areas where the movers and shakers are
really making a difference.
Well, thank you for joining
us this afternoon or this morning,
wherever you may be, and,
Cindy and Sarantis,
till next time, so long.
Thank you
for listening to the Proteomics in Proximity
podcast brought to you by Olink
Proteomics. To contact the hosts
or for further information, simply
email info@olink.com.