Howard Jacob: Thank you very much and thank you, everyone,
for being here. The goal of this is really to capture some thoughts as we go forward
with the day. So, the overview -- so, obviously GWAS and clinical sequencing are changing
how we practice and will practice medicine and research. I think that’s clear, but
what’s the levels of evidence that is required as we move down the road? So, we can use traditional
QTL to gene in animal models, or from QTL to gene using GWAS, from GWAS to gene variance,
and obviously with testing variants of uncertain significance.
So, we have a lot of expertise around this, and I just want to walk you through one example
that’s happened in our particular case. So, we had a patient that we were actually
studying from the chronic kidney disease consortium, and this individual patient had a mutation
in Shroom3, and this particular variant was a variant of unknown significance. It’s
highly conserved down through a whole bunch of different beasties [spelled phonetically],
and so the question I want you to think as we go through this is what data would you
require to say this variant causes chronic kidney disease to put in a medical record,
okay? So, that’s really the threshold I want you to think about, not to write a paper
but to put in a medical record. There’s not going to be in most of these cases randomized
case control studies to get these variants out there. So, that’s what I want you to
think about as I go through this talk. So, Shroom3, so what do we know about Shroom3,
well, it’s a very commonly discovered GWAS hit. It’s one of the most reproducible.
Its function is unknown. There’s over 11 GWAS that have been hit, and it’s been associated
with glomerular filtration in albuminuria, and it’s associated with all populations.
So, it seems like a reasonable candidate. However, the evidence in the biology at the
time when we got involved in this was it was known nothing about what it was in the kidney.
There was basically no information and in fact the homozygous null mouse was lethal.
So, you didn’t have the ability to go walk into a mouse and ask a simple question around
that. So, this is where we found ourselves.
Now, the reason we got involved in this is really because of rat data we’d been doing.
We’d done QTL mapping over many, many years. In fact, this was a project that I had started
with Lander when I was a posts-doc with him a long, long time ago, and so shoom3 happens
to sit in the QTL in chromosome 14. So, there’s evidence around that, and we went in and took
a look at this from the fawn hooded rat, which was the disease model, and the brown Norway
rat, which is the control. And this is the characteristics. There are glomerular hypertension,
proteinuria, focal glomerulosclerosis, and podocyte effacement really characteristic
of what you’d find in the clinical environment. And we had taken it down and actually found
this particular gene here, Shroom3, and all of the red variants are variations for which
he would say are potentially dysfunctional. So, there’s multiple variants in this gene
that you would predict will cause this protein to break. Okay, so the interesting part about
that, well how would you attack that? Well, what we did was we then used zebrafish and
we went in, and then modeled this. And so, what you have to understand is that for renal
function, the green dye should stay. So, when the kidney is working normally, you just keep
the green dye, and when you have a dysfunctional podocyte, what will happen is you’ll lose
the green dye, okay. So, that’s the main element. So, if you put a morpholino in for
Shroom3, so you knock out the zebrafish, you actually see that it goes away. If you now
put in a normal rat variant, you will now see that you can actually recover the phenotype
completely. So you take out the zebrafish and you can replace it with a rat. You now
then can show that if you now take these three different regions, and we then took these
different regions, and substituted them into the zebrafish genome for these genes. And
what we were able to do then was narrow which part of the rat actually had was functional.
And what we found using the zebrafish as an assay was that these potential variants were
now causal. So, we started with rescuing and being able to narrow it down. And we actually
got it down to the absolute variant that was causing this disease if you will in the rat.
So, with this information it was GWAS nominated. There’s QTL data in the rat. Interesting
about this is this one QTL in the original mapping, this gene actually is -- there’s
two genes within that same QTL. So, in the paper there’s the -- the nuance is around
that, but the point is that it’s functioning [spelled phonetically]. We know what’s causing
it in gene editing in the zebrafish we used to [unintelligible] the cause of mutation.
So, how many people in this room would be willing to write in the medical record now
-- obviously some of you aren’t M.D.s, but you can pretend you are, would put in the
medical record that this is now enough evidence to say that this gene causes renal failure?
Okay. One, all right. We’ll move to the next.
[laughter]
So, how about more zebrafish data? Can you take this to more function, because really
I just told you that this variant has a broad effect, right? So, what we’d like to be
able to do is take this into a deeper depth, and so what we did then is we used the zebrafish.
And now what I want to talk about is being able to go in and look at podocyte effacement.
That’s one of the key characteristics. So, zebrafish although it’s a pronephron does
have glomerular filtration membrane, and does have podocytes which you can see listed on
the slide here. So, we then went in and did a series of studies that I just showed you
before looking at the green florescence protein labeled, and seeing what was happening. Sorry,
this was dextran [spelled phonetically], and so here we went in. We did the same study,
were able to show that we can cause skipping. We were able to go in and show that the Shroom3,
we can knock it out, and we can show that it has function. So then, what we did is we
did a really interesting strategy. That’s just to tell you how this works. Hypothesis,
that it regulates the filtration barrier. So, this is the strategy. So, we then went
in and we now used a GAL4 mutation. So, we got this variant from Ian Drummond [spelled
phonetically], and we’re now able to go in and take a podocyte specific and be able
to say, “Okay, we’re attaching it to pitocin. And now, we’re only going to express and
be able to knock this out specifically. So, now we’re showing [unintelligible] you express
this gene and it’s broken, it should cause podocyte effacement. And sure enough it causes
podocyte effacement. And it showed -- we can show also in the proteinuria. So, now we’ve
been able to say, “Okay, it’s not just this particular variation, but we can take
it all the way down to showing that there’s podocyte effacement, which is actually what’s
thought around this.
So, now we’ve added more information. So, now that we can show that in zebrafish that
there’s podocyte effacement, how many of you now would say that this is what causes
the gene in humans?
[laughter]
Okay, could cause -- okay, would write in the medical record? Let me say that. Stay
with the same question. How many besides Terry [spelled phonetically]? Anybody else would
add the -- we got one more person, okay. A few more people, okay. All right.
Female Speaker: But this patient already has kidney disease.
Putting it in their medical record --
Male Speaker: Right.
Female Speaker: -- is not such a big deal. It’s how you’re
going to interpret it for the future.
Howard Jacob: Keep that in mind. That is a position, okay.
So, keep that in mind. How about if we just test the patient’s variant? Wouldn’t that
be a really good idea? So, that’s -- guess what. We did the exact same strategy, and
we now took this patient’s exact variant. And we showed what I showed you before. And
now, what we did is we went through and we actually went in, and we took a common variant
from a human. And showed that it rescued. So, we rescued the phenotype, and we made
the exact single base pair [spelled phonetically] mutation from this patient. And showed that
it caused the disease in the zebrafish, okay.
So, same argument. Would you write this in the medical record? Okay, so we’ve been
through this exercise. Now, let me tell you the interesting part about this. I presented
this at the American Society of Nephrology in November, okay, and I asked this exact
question. So, of 500 clinical scientists in the room, how many of them do you think agree
that they would write this in the medical record for this patient with this data?
Male Speaker: Zero.
[laughter]
Howard Jacob: Zero. Zero, and in fact what I got were four
people that stood up, that told me that “A zebrafish is not a human. A rat is not a human”,
and there’s now data that’s also come out that I’ll show you next, that “a mouse
is not a human.” And so, it doesn’t matter how many times you do this without a randomized
case control study, we are not going to write this in the medical record, okay. So, the
reason I’m telling you this story is that this is a group of people who were trying
to figure out how to do this. Now, there’s a lot of education and the point of this is
really to illustrate what people think. This is a paper that just came out. I don’t think
showing that a mouse would either convince people.
So, the point that I want to make, the simple point in setting the frame of this meeting
is I think that in this room we think about sequence first, ask questions later is going
to ask a lot, drive a lot of basic research. I think basic research at the speed of the
clinic is going to be absolutely essential, but how do we define proof? Now, obviously
there’s risk benefit. The sicker the individual the more were willing to do this, but as we
walk into this meeting I just want you to think about that if at least one perspective
if we’re going to try to effect care, the data upon which we’re trying to drive needs
to demonstrate enough evidence, whatever that is. And so, that was the only point that I
wanted to make. There’s a lot of people that were involved in this. My former laboratory
up at the Medical College of Wisconsin, Flo [spelled phonetically], actually this was
her thesis work. She’s now a post-doc with George Church [spelled phonetically]. A large
number of people that I’ve worked with across the team, and then also the chronic kidney
disease. So, I’m happy to take questions. I’m not sure there’s a lot of questions
around this, but I’m really more about setting the stage of where we’re going for the rest
of the two days. Thank you.
Male Speaker: Thank you, Howard. So, Rex first. Mark next.
Nancy next.
Nancy Cox: In just a few.
Male Speaker: Les next.
Male Speaker: You’ll be happy there aren’t any questions,
right Howard?
[laughter]
Howard Jacob: Yes.
Male Speaker: So, I think this is a really great way to
start off this meeting, because it really does highlight what it is that we’re trying
to think about. In the minds of those 500 people in the audience, you know, how do we
ever get over this idea that you need some kind of a randomized control study in order
to do that, because that’s going to be a barrier that we’re never going to achieve?
So, thoughts?
Howard Jacob: I have to be completely honest. So, I was
shocked, okay. I didn’t expect a huge number of people to actually stand up and agree with
us, but I was shocked that nobody was willing to raise their hand that they would write
it in the medical record. As Nancy said, hey, this is a patient. We already know there’s
a mutation that’s there. You’ve gone through all of this elaborate biology. I expected
some agreement around that. So, I honestly don’t know, and that’s why I think this
meeting’s incredibly important. Now, I’m a Ph.D. So, I have a different mindset on
this, and I think that’s why this is really important to have the physicians in the room,
and the basic scientists around the room, because if we can’t convince the physicians
here that I’m going to argue or are more willing to think about it, then I think we
can’t take it out to the general population. But that’s just an opinion.
Male Speaker: So, my question was related to what I consider
to be a huge pile of missing data that as a clinician, I would need to understand to
be able to interpret the functional data, and that is what’s the allele frequency?
Has this ever been seen in any of the exome collections? Is there any evidence of segregation
data? You know, are there other individuals that have been reported with different mutations
in Shroom3? Those are the types of things, and it may be a null set, but I think you
know, with -- absent any of that other data, the functional data while convincing would
certainly not be enough for me to be definitive about causation. I might suggest, you know,
that we could put something in there to say, “Well, there’s a variant in this that’s
of uncertain significance. It needs additional study”, but if you think about the implications
of making that assignment since presumably this is now something that you’re proposing
as [unintelligible] dominant, now you’re thinking about cascade screening and all the
other impacts of this. And so, those are the types of things that I need to be able to
contextualize that I can’t make a decision based solely on functional data.
Male Speaker: Short answer, Howard, and then we’re going
to stop.
Howard Jacob: Yeah, so for the sake of clarity, and which
I didn’t get into this, so chronic kidney disease is a polygenic, multifactorial disease.
And so, in this particular case there is evidence in the population of Shroom3. That was the
GWAS data. So, the challenge on this is a little bit harder than what we’re talking
about for the Mendelian disease, and that it is in fact only one of the elements. It
is no way the only element, and it will be entirely possible. And I’ll even predict
that in the cohort there are going to be people that have a mutation in this gene, that do
not -- that get the disease because there’s complimentary. So, the complexity of this
is a little bit more, because it’s a common complex disease. Common in the sense that
there’s more than 200,000 people that have this, but even with that information. So,
I think those are fair points and thank you. I think that at least stimulates I hope the
conversation over the next few days.
Male Speaker: I think we could probably sit here and talk
about that presentation for the rest of the day.
[laughter]
But we won’t. So, Monte Westerfield will give the basic science perspective on need
for integration, and if you want to know anything more about him, read it in the biography.
[end of transcript]