- Welcome everyone to Wednesday Nite @ The Lab .
I'm Tom Zinnen, I work here
at the UW Madison Biotechnology Center.
I also work for UW Extension Cooperative Extension,
and on behalf of those folks, and our other co-organizers,
Wisconsin Public Television,
the Wisconsin Alumni Association,
and the UW Madison Science Alliance,
thanks again for coming to Wednesday Nite at The Lab.
We do this every Wednesday night, fifty times a year.
Tonight's rather special, because this is a Wednesday night
about the lab, and this is about the Labrador Retriever,
and a particular type of degenerative polyneuropathy
that that breed and other types of dogs get.
Our speaker tonight is Susannah Sample.
She's with the School of Veterinary Medicine.
She was born in Hinsdale, Illinois,
and grew up in La Grange.
She went to Lyons Township High School,
in the suburbs there.
Went to Northwestern University and got her undergraduate
degree in biomedical engineering.
Then she came up here to Wisconsin,
where she got her Masters, her DVM, and her PhD,
and then, just last year,
she became a board certified veterinary surgeon.
So, I'm looking forward to hearing about
one health medicine, a naturally occurring dog model
of degenerative polyneuropathy.
Please join me in welcoming Susannah Sample
to Wednesday Nite at The Lab.
[audience applauds]
- Well, thanks for that introduction.
To go through a quick outline of
what I'm going to talk about tonight,
I was going to start by discussing a little bit
about canine genetics, and genome wide association studies,
followed by a discussion about this condition
that I'm studying in dogs
which I call acquired laryngeal paralysis polyneuropathy,
some people would just refer to it as Lar Par,
more commonly.
And I'm going to go through a few basic definitions
about genetics and genomics to get everyone on the same page
to explain what we're doing and why,
and then discuss our genetic study,
and a little bit about future directions.
So to start, when you talk about
canine genomics and genome wide association studies,
dogs have really become more prevalent in this area,
and so I'm going to talk a bit
about why dogs are really great models.
In 2010, there was this paper that came out with the title
Man's Best Friend is Biology's Best in Show,
and I just think that that's a really great encompassing
title to talk about the idea that dogs are really important
when it comes to comparative genomic research,
and the reason for this, is that when you talk about
pure bred dogs, they are a very highly inbred
group of animals.
Selective breeding has created over 400 breeds,
and most of this has happened within even
just the last few hundred years,
and as a consequence of this, we have a very high
prevalence of breed specific diseases.
Ultimately, this really, when you think about it,
is humanity's greatest genetic experiment.
It started, you know, thousands of years ago,
when we first took gray wolves and domesticated
them into what's referred to now as village dogs,
and over the last few hundred years,
taken those dogs and created dog breeds
to the extent that you end up with, you know,
situation where you've got this little guy
and a Great Dane, that are actually the same species,
and when you really sit back and think about that,
it's a pretty amazing phenotypic
or visual diversity within dogs.
So, importantly, when you think about canine genomics
or genetics, the canine and the human genome
are actually very similar.
So, at this point, about 92 percent of genes
that we know of in people have been mapped in dogs,
and that's obviously increasing over time.
Another important feature is that people and their dogs
share a common environment, so environmental influences
are obviously quite similar,
and most diseases that we see in dogs
do have a corresponding disease in humans.
So, talking about one of these diseases is
acquired laryngeal paralysis polyneuropathy.
So like I mentioned, in the world
a lot of people call this disease Lar Par,
and it's most commonly what we call
an idiopathic degenerative polyneuropathy.
So, what that means, idiopathic means
we have no idea what causes it.
Degenerative means, obviously, it's something
that degenerates over time,
and polyneuropathy means a disease of many nerves.
About 70 percent of these cases occur
in Labrador Retrievers.
The next most common breed, at least in the United States,
are Golden Retrievers.
Rarely, dogs can get laryngeal paralysis from other causes.
Most commonly, that's going to be from a tumor,
maybe in their neck, or from some sort of surgical trauma.
Technically speaking, endocrine diseases like
diabetes or hypothyroidism could cause a disease like this,
but that's not really been well-documented.
In Labrador Retrievers, the age of onset
or presentation is very typical.
So, they tend to be about 11 maybe 12 years of age,
and owners usually report that they've noticed a change
over the past year,
and it strongly resembles certain neuropathic
conditions in people, so the example I'm going to use
tonight is a disease condition called
Charcot-Marie-Tooth disease.
So, talking a little about Charcot-Marie-Tooth,
it's the most common inherited polyneuropathy in people,
or peripheral neuropathy in people,
and what that means is that it's a disease of nerves
that aren't necessarily coming in your spinal cord
or your brain, but they come from those places.
So they're what's innervating your body
from your cord or from your brain.
And it's actually, relatively common.
It affects about one in 25 hundred
people in the United States.
It ends up having a progressive degeneration
of motor and sensory nerves,
so these people tend to experience muscle atrophy.
They have chronic pain.
They have hand and foot deformities,
like you see in that picture there.
And interestingly, and importantly,
it's the longest nerves in the body
that tend to be the most affected.
So, in people, those nerves are usually
what innervates your distal limbs,
so your hands or your feet,
obviously progressing over time.
There are a lot of different subtypes,
so when you think about Charcot-Marie-Tooth disease,
you have to realize it's an umbrella term.
So, it's a disease term for a typical presentation
that is caused by a lot of different genetic mutations.
So, I may have CMT, and someone else may have
CMT in the audience, but what's causing that
would be two different genes.
So, at this point, there's over 80 genes
that are associated with it.
Now, there are some that are a lot more common than others.
So, there's four genes responsible for most
of the genetically diagnosed versions of this disease,
but not all cases have been found--
We haven't found a genetic cause for every type of case.
The other important part of this is that
most forms are inherited in an autosomal dominant fashion.
That's not always true,
but the vast majority of them are a dominant disease.
There's no disease modifying therapy currently available
for people with this condition,
and so the treatment ends up being symptomatic,
so we help to support people with disabilities,
or you treat muscle cramps, or spasms, or pain.
One of the big concerns you have
with any time you are dealing with a neuropathic disease
is that when you talk about drug trials
there's obviously a lot of ethical concerns
that come up about the idea of trying drugs
that affect your nervous system,
particularly when you're dealing with children.
Now, Charcot-Marie-Tooth can affect children,
but generally, it's thought of as being a middle-age
to older disease because it's progressive over time,
so people tend to start having signs
that really affect their life when they're a bit older.
And like I said, not all genetic underpinnings
are identified, so one question we have is
is this disease we talk about in dogs,
a potential model for a condition in people.
So, let's talk about laryngeal paralysis.
Like I said, when you look at this clinically
you can see signs of a peripheral polyneuropathy,
so again, nerves not from, not your cord or your brain,
but nerves coming from those places,
that are affecting those nerves
that are longest in the body.
Now, in the dog, the two longest nerves you have
are the recurrent laryngeal nerve,
which, I have no idea why, but it's true in people too,
this exits your brain, it comes down,
it wraps around your heart,
and then comes all the way back up
to innervate your larynx.
So, in a giraffe, this thing's like eight feet long.
It's the most redundant nerve ever.
And the other long nerve you have is the
nerves in dogs that innervate their hind limbs,
those are another very long nerve.
So, when you look at these clinical findings,
dogs end up with laryngeal paralysis,
which is their larynx, or voice box is paralyzed.
They can end up with respiratory distress.
Their esophagus isn't going
to have correct mobility anymore.
And you end up with problems in your hind limbs too.
So, clinically we see hind limb weakness
and decreased reflexes in the back leg.
So, I'm going to talk a little bit more
about all of those in a moment.
But clinically, when people come in
with their dogs having this disease,
the first thing to realize is that dogs
don't ever exhibit symptoms, they exhibit clinical signs.
So, what that means is a dog can't tell you
if when they're eight, if they're feeling tingly
feelings in their paws, right?
They can't tell you if their stomach hurts,
or if they're nauseous.
Instead, they vomit, right?
So they show you the clinical sign
that they're not feeling good,
but they can't tell you, you know, three hours beforehand
like, I'm not really feeling so good.
So, consequently, we don't really have a way
of identifying this potentially earlier on
until there's really a major problem,
where these nerves aren't working at all.
The typical progression when owners report in Labradors,
is usually relatively slow from months to years,
they start noticing a few changes.
And usually it's the changes associated with the voice box,
or the larynx, are the most noticeable.
So, oftentimes dogs have a voice change,
or instead of barking normally,
they have a very hoarse bark.
Maybe they won't want to exercise quite as much.
They end up with very loud breathing,
and in very severe situations,
they can end up in extreme respiratory distress.
They can turn blue, which we call cyanosis,
and potentially even collapse.
Hind limb weakness is also often noted
when you start talking to owners about it,
but unfortunately it's often misinterpreted
or misdiagnosed.
These are older dogs, so the hips get blamed a lot.
That they have hip dysplasia, or old-dog arthritis,
or they're just slowing down,
when really, they have a neuropathy
that's affecting their mobility.
On examination findings, you see
severe upper respiratory noise, and like I said,
some neurologic exam findings, you can find.
So, I'm going to go through all of these.
They have absent paw replacement tests.
So, I'll show you what that means.
Decreased withdrawal reflexes,
and I'll discuss that and then also show you
some images of a dog with hind limb weakness.
So the severe upper respiratory distress,
or noise and potential respiratory distress,
is something that you can generally hear
these dogs coming down the block.
They sound something around Darth Vader slash train.
And so, I'm going to play a video of this,
and you have to realize that there's a dog--
It's not like this dog's just been exercising.
He was just sitting down and stood up.
So, you get a sense of how noisy he really is.
[noisy panting]
He's very excited.
Shake it off, shake it off.
Shake it off.
So, you know, later on in the disease,
these dogs are actually relatively obvious
when they have the disease, 'cause you can hear it.
Other things that you see are,
laryngeal paralysis on exam.
So, these are images of a larynx
and they're taken through a small camera
that we actually use to look inside abdomens of dogs,
so the resolution's not great, and I apologize,
but, to get you oriented,
we're looking down the throat of a dog.
So, up here, is the esophagus.
That little crease right here.
This is the trachea inside that dark hole,
and these are cartilage
that is associated with the voice box.
This is a tongue depressor.
So, to give you a sense of a normal dog,
when they breathe in, you'll see that--
Whoops, that's not it.
When they breathe in, the larynx opens, right?
So, normally when you breathe in,
these sort of double doors open up, and then close.
So, I'm breathing in, I'm breathing out.
I'm coughing, I'm breathing in, I'm breathing out.
So, in a dog that's paralyzed,
I don't know how well you can see this, but,
there's a little bit of mucus right here,
and so you can see that move when the dog breathes,
and you'll notice that the larynx is not moving at all.
So, he breathes in and nothing's really happening.
And that's all to do with this recurrent laryngeal nerve.
Another sign we see, are the paw replacement tests.
So, normally, if you take the hind limb of a dog,
and their paws, and you flip them over,
they should flip right back.
So, dogs that have this condition,
oftentimes, don't do that.
So, you can see that this dog,
if you flip his paws over, it's just not really connecting
that that's happened, and they don't flip them back.
Another sign, and this is a little hard to see,
but it's decreased withdrawal reflexes.
So, I'm going to show you a normal dog,
and then I'm going to show you a dog
that has decreased reflexes.
So, this is a dog that's--
A withdrawal reflex.
So, what I'm going to do first with this dog
is show you what a normal range of motion is,
So this is what he should do.
You pinch his toe, he should pull back,
and all of his joints should flex.
So, you can see that his ankle flexes,
his knee flexes, and his hip flexes.
It's the same reflex that if you touch a hot stove,
you pull your hand back.
That's not something that goes all the way up to your brain
and back, that's a local reflex,
and if you ever have the opportunity to do this,
you'll notice that your wrist, your elbow, and your shoulder
all flex back, so that's normal.
So, in a dog that has this condition,
what ends up happening, oh--
Is they go away.
There.
So, I'm going to again, I'll show normal,
so that's how he should flex his leg,
and what you'll notice is he kicks back
when you pinch his toe.
So, he doesn't flex this joint correctly.
He's really not flexing it.
The computer's touchy.
And he kicks back.
And that's because his hock's not innervated,
or his ankle isn't having normal innervation.
Lastly, you start seeing, hind limb weakness.
So, when you watch a dog walk normally,
you can notice that, he's trotting
in a very sort of graceful way.
He's flexing his joints correctly.
When he puts his feet down,
they're not totally under his body,
but they're in an appropriate place.
That's really a normal trotting dog.
Now, when you see a dog that's weak,
what you'll notice,
is that he walks in a very stiff-gaited manner.
He's not really flexing his joints correctly.
He's maybe a little bit more of a wide based stance,
and that all just has to do with him
being relatively weak.
So, these are very classic signs
that we can see on neurologic exam.
Unfortunately, this can often become an emergency.
Particularly when you have owners
who just aren't aware that their dog has this condition.
They don't appreciate that maybe these things
aren't just old dog changes.
This particularly happens when dogs are excited,
and it's high temperatures.
So, their respiratory rate goes up, they start panting.
That airway flow, and flow of oxygen going by
actually causes some trauma almost to the laryngeal mucosa
which is just like the cheek, inside your cheek,
so you can imagine it swells relatively quickly.
It becomes inflamed, starts to swell,
the dog's aren't breathing so well.
Now, they're really upset, and you end up
in this sort of vicious cycle,
which can, in very severe cases, end up
with what we call severe dyspnea, in other words,
a very difficult time breathing.
They can end up with cyanosis, they turn blue
because they're not getting enough oxygen.
They can collapse.
And because dogs cool themselves so much panting,
they can end up with hyperthermia,
and actually die of heatstroke.
So, unfortunately, this really is a life limiting condition.
The most common problem we have with dogs
that die of this disease is that
they get aspiration pneumonia,
and what that means is essentially
food goes down the wrong tube,
and whether it's food going in, or actually most likely,
what's more likely is their actually getting regurg,
or esophageal reflux, because their esophagus
isn't working quite right,
and now, their larynx, which protects
their airway isn't working, and you can imagine,
that they can end up with what we call silent aspiration.
So, they can end up with pneumonia.
Sometimes, that can be catastrophic to the point
where there's really not much we can do,
and sometimes it's something that just happens
over, and over again.
And you have these older dogs,
that are in the hospital a lot,
and they're not able to live their dream.
On the flip-side, you can have severe laryngeal swelling,
like I talked about, where you will have dogs
that die of heatstroke, or you have dogs
that die literally of suffocation.
And sometimes, what ends up happening,
is these dogs just degenerate to the point
where they don't have the quality of life
that they want to have.
They can't chase their squirrel, you know,
they can't go do the things that makes them
wake up every morning, and owners just say,
he's not doing what he needs to do to be happy.
So, in terms of treatment options,
there's, like Charcot-Marie-Tooth,
there is no disease modifying treatment available.
We have two options.
We have conservative management,
and then we do have one surgical option.
So, conservative management usually involves
weight loss to keep these dogs from getting so hot.
Stress management reduction so they don't get too excited.
Exercise restrictions, obviously, so they don't end up
panting so much, and avoiding high temperatures.
Surgically, we can go and open up their airway
to a degree, which essentially gives them
sort of a rescue tube to breathe through,
so hopefully they'll never asphyxiate, or suffocate,
or they'll never have a heatstroke event.
And there's a number of different ways to do this,
and if you put ten surgeons in a room,
you'll probably get eight opinions
about which is the best.
But generally, what's done, in 2016,
is a cricoarytenoid lateralization,
which is commonly referred to as a tie-back surgery.
There's other options as well,
but what it essentially does
is if you look at these images here.
So, we have the larynx again, up here is the esophagus.
Trachea again is here.
So, if you look at this opening in the trachea,
what these procedures essentially do,
is open that up to a degree,
so you can see that this area,
which we call the glottic opening, is a little bit bigger.
And it just gives them a rescue,
so, they don't end up in those really dangerous situations.
We talk about prognosis with these surgeries.
Aspiration is a complication.
And if you read through the literature of
different lateralization procedures, or opening procedures,
on average, somewhere around 15-20 percent of dogs
probably get aspiration pneumonia after the procedure
in the immediate, sort of, few days.
Now, not very many dogs die of that,
they just need to be in the hospital a little bit longer.
But you can have catastrophic aspiration events,
as a result of this surgery.
They're very rare, but as soon as you see one happen,
it makes you nervous.
Unfortunately, we have no pre-operative means
of determining which dogs are going to get
aspiration pneumonia post-operatively,
so there's not really a good way to recommend to owners
whether or not this surgery is something
that's going to be more dangerous
for their dog versus another.
But you also need to keep in mind,
that aspiration pneumonia is also common
in dogs that don't have this surgery,
so it's kind of a complication of the disease in general.
And these are two images of a thorax of a dog
that shows you aspiration pneumonia.
So this is a normal dog.
So, when you're looking at this, the head's this way.
His little butt's that way.
This is his heart.
So, you're looking at his lungs,
and this is his trachea coming down.
And that's really a normal thoracic radiograph.
When you look at this dog, what you can see
is that there is this area that is white,
and what looks almost like a tree coming out of that,
and those are what we call air bronchograms.
And essentially what causes that
is when there's fluid in the lungs,
the bronchus, which still has air in it,
contrasts very well on these radiographs,
and you can see sort of this tree-like pattern
that occurs and it's a very classic radiograph
of what we call aspiration pneumonia, or any pneumonia.
So, the key points here is that
laryngeal paralysis in dogs really
is a devastating disease.
It can be life-threatening.
The clinical features indicate
that it's a peripheral neuropathy.
It's similar to a number of human degenerative
neurologic diseases, one of which
I discussed with you today,
and it really does have a potential to be a naturally
occurring animal model of human peripheral neuropathies.
I'm going to take a quick drink.
This is one of the dogs in our study.
He's very handsome.
And apparently a winner.
[audience laughs]
Alright, so I'm going to talk a little bit about genetics.
So, stick with me.
I'll try to make this as painless as possible.
So, we're going to go back to like fifth grade, and DNA.
So, DNA is the inheritance information of your body,
and each strand is essentially made up of four bases.
So, A, G, T, and C.
And each base, essentially creates
what's called a nucleotide.
So a given nucleotide would be like, A,
and when you match A with its pair, which is T,
you get a base pair.
So, A always pairs with T, C always pairs with G.
That's the basic you need to know about
the nucleotide, the base pair, and DNA.
Secondly, the genome.
So, the genome is the entire set of DNA for any animal.
You have to realize that 99.9 percent of it is
exactly the same in a given species.
The caveat of that is point one percent
is actually a lot of DNA, because the DNA is huge, right?
[coughs]
So, when you talk about DNA,
oftentimes, we'll discuss alleles,
and alleles are alternative forms of DNA
at a DNA segment at a given location in the genome.
The way I always like to think of this conceptually
is that, from your mom and your dad
you get two alleles right?
So, you've got one from each parent.
When I refer to the word locus, which I might slip into
every once in awhile, that essentially
just refers to a place in the DNA.
Alright, it's like a house, a location, a locus.
Okay, I promise this is the last one.
Genotypes versus phenotypes.
So a genotype is a pair of alleles at a locus,
and so you can be either a homozygote or a heterozygote,
and a homozygote is when you inherit two identical version
of the allele from your parents,
or heterozygote is when you inherit two different alleles,
and the phenotype is the observable characteristic
as a result of the genotype.
So, just to bring back freshman year in high school,
a great example of this is eye color.
So, when you look at brown eyes versus blue eyes,
you probably all like drew this chart back in the day.
You can either be a homozygote major,
where you're big B big B, and you get brown eyes.
Brown eyes being a dominant trait.
You can be a heterozygote, where you're big B little b.
You end up with brown eyes, because brown is dominant.
Or you can be a homozygote minor, which is the minor allele,
which is little b little b, and you end up with blue eyes,
because blue eyes is a recessive trait.
So, now that we've bought that flashback,
let's get into something more fun.
So, genome wide association studies--
the entire point of this is to come up with a way
to scan the genome and find a biologically significant
variations that contribute to a trait or a disease,
and when you think about this,
it's really a very nascent field.
So, it wasn't even really thought of until the mid 90's.
The human genome wasn't sequenced until 2001.
It wasn't until 2005 that the first human GWAS came out.
That same year the canine genome was sequenced,
and in dogs, our first GWAS came out six years ago.
So, this is really um, a relatively new field,
but it's very rapidly evolving because it's been,
I think realized, that it's a very powerful technology.
So how this works.
I'm doing to talk about SNiPs and LD
in more detail in a minute.
But, essentially, there's commercial arrays out,
we call them SNiP chips,
that have a whole bunch of these SNiPs scanned across
the genome, and you send in DNA for your SNiP chips,
and you get a whole bunch of data back,
and you can analyze them,
and there's two real ways to approach GWAS.
You can either do a case-control association,
like I'm doing, so the dogs either have laryngeal paralysis
or they don't, or you can do quantitative trait association,
so for instance height.
So, there are some genes that are more correlated
with being tall, and other genes that are more correlated
with being short.
And the power of a SNiP to detect an association
is dependent on something called linkage disequilibrium.
So SNiPs in a simplified fashion.
They're variations in a nucleotide base
at a single locus in the genome.
So, in one spot in the genome, there are variation
in this A, C, T, and G, that's recognized within a species.
So, if you look at this here.
Essentially, this is, say, a strand of DNA,
and here is three subjects, and here's a bunch of SNiPs.
So, essentially these arrays take a bunch of SNiPs
and they put a scan throughout the genome,
and I'll get back, say three subjects,
so at SNiP one, this subject is an A-T,
and this subject's an A-A, and this subject's an A-T.
And then obviously you have that,
except for a few hundred thousand, or a million SNiPs.
Some of these SNiPs are biologically significant,
and some of them make absolutely no difference.
So these studies, essentially, use these SNiP markers,
and like I said, they're just spread out across the genome.
So what about linkage disequilibrium, what does that mean?
So, when you think about linkage disequilibrium,
the easiest way to conceptualize it
is to realize that there are chunks of DNA
that are inherited together.
It's easiest to think about this in a linear fashion,
like DNA's one big strand, and there's big chunks
that are inherited.
In reality, sometimes these aren't
even on the same chromosome.
Sometimes, they're pretty far away from each other
on a given strand of DNA,
but when you're thinking about it for now,
just think about 'em as a chunk of DNA
that's all inherited together.
And you have very long chunks of DNA in new populations,
and shorter chunks in old populations,
and we'll talk about that in a moment.
The other thing to keep in mind,
is that pure bred dogs are not bred randomly, right?
There's nothing natural about a stud dog being bred
400 times in its life, so,
the lifetime matings for given dogs can be very high,
and it just really highlights the idea that
pure-bred dogs, their genetics, and breeding
are very controlled.
Alright, so this is the tough bit.
These are called triangle plots,
and they're really the easiest way to describe
linkage disequilibrium, and the way to think
about this, is it's like a heat map.
So, along this top area here are a bunch of SNiPs.
So, RS is a common, and then a bunch of numbers behind it,
is a common way of identifying a given SNiP.
And so, within each one of these,
what you can do is connect them, so,
here's SNiP number three, and we're going to connect
it to SNiP number nine, and you can see,
because it's sort of a reddish color,
those two are in some degree of linkage.
In other words, they are often inherited together.
Alright, so the more red you have within these plots,
the more linkage disequilibrium exists.
So, to put that into context,
when you look at human populations,
you can see newer populations, like Caucasians,
which in the history of humanity,
Caucasians are actually a very young population,
versus an ancient population like the Yorubans,
and when you look at the difference in these maps,
you can tell that the Caucasians have a whole lot more red
than the Yorubans.
So, newer populations have higher linkage disequilibrium--
In these heat maps, they have more red,
because more of these chunks of DNA are inherited
together, and associated with each other.
So that's people.
So what about in dogs?
Well, when you look at a Caucasian,
you're like, yeah, there's a lot of red there,
and then you look at a pure-bred dog,
and it's like red-out, right?
So, pure-bred dogs have extremely
high linkage disequilibrium, and that becomes important
when you're doing genetic studies,
because within one of these blocks or chunks of DNA,
when you're looking at SNiPs and you're trying to find
a disease, you only need, theoretically,
one SNiP within that block to tag a disease locus.
So, within here, if you're saying, here's my disease,
so in humans the disease is here, and in dogs it's here.
In order to find that disease in people,
because they have smaller linkage disequilibrium,
because their DNA isn't inherited in as big of chunks,
you need all of these markers to try to find
that particular mutation,
versus a dog, because you have bigger chunks of DNA,
and you only need one of these markers in each chunk,
you end up with needing a whole lot less markers.
And this is really reflected in these BeadChips,
of SNiP chips that I was telling you about,
that you can get commercially.
So, in people, their SNiP chips have
over 4 million SNiPs across the human genome versus dogs--
we're like really excited because
we just got bumped up to 220 thousand.
So, when you're looking at that,
it really just gives you a sense of how fewer
you know, markers you really need to look at this in dogs.
So, the key points of this is that
genome wide association studies are a means
to scan the genome for variants that associate
with a given disease of a given trait.
And the power of GWAS is really influenced
by linkage disequilibrium, so dogs have a very high
linkage disequilibrium compared to people,
and what this means is that when you're studying
diseases in dogs, you need fewer sample sizes,
you need fewer dogs enrolled, and you need
a lot less markers across the genome
to try to find a problem.
This is Botchy.
He's fabulous. Okay, so--
I'm now going to talk about
Mendelian versus complex traits.
I promise this gets exciting in a minute.
[audience laughs]
Wait till you see the videos.
So, when you talk about Mendelian disease,
these are diseases that you either have the gene
or you don't, so on this sort of map here,
and you're saying, well what gives me this disease.
Well, Mendelian diseases are pretty much,
your genetics gives you the disease,
versus an infectious disease, say Ebola,
where like, it's in the environment,
you kinda got it regardless of what your genes are.
And sitting in the middle of this,
are what's called complex traits.
And complex traits are diseases that
you may have genetic susceptibility to it,
but you also probably have environmental factors,
and other factors, that have,
sort of tipped you over the edge
to getting the disease.
So, an example of this is Alzheimer's.
Right, so, there's no genetic test for Alzheimer's.
There's some genes that may predispose you to it
more than others, but it's complex,
there's a lot more going on.
So, this is my video.
So, wait for it.
So, this is our dog, and right now
he's on the scale of being disease negative, alright?
So, he has no disease.
So, when you're talking about Mendelian diseases, right,
you have a gene, and it gives you the disease.
So, that mutation just pulled him over,
and now our dog is diseased.
When you talk about complex diseases,
which are a little bit more difficult,
essentially you have a whole lot of genes
that are kinda contributing together,
in sort of an additive effect, or Skittles,
depending upon your perspective,
and they contribute together to create a situation
where you have a disease,
but that's really not totally accurate.
Because, if you're a dog, you may have all those Skittles,
and all those gene mutations that are putting
you in a position where you are susceptible
but there's going to be something in the environment,
e.g. milk bones, that might
tip you over to getting the disease, right?
So, maybe obesity, or other environmental factors
that really contribute to you getting that.
So, it's really the difference between monogenic
or Mendelian traits, and complex diseases.
When we think about Mendelian disease,
there's a number of examples of these in people,
like cystic fibrosis, or Huntingdon's disease.
There's a paper that came out in 2015,
looking at canine idiopathic epilepsy,
which is a seizure disorder and idiopathic again,
meaning we don't know what causes it,
where they used--
whoop, getting ahead of myself--
They used 157 cases and they had 179 controls.
That may seem like a lot,
but you have to realize that if this study
was done in people, you'd have thousands,
and we'll get to that.
And essentially, they ended up doing a study,
and what I want to talk about actually here
is what this whole plot it,
and so, it's called a Manhattan plot,
and I think it's because it's supposed to look
like the Manhattan skyline,
but each one of these little dots here is a SNiP.
And along here, you've got your chromosomes.
So all these SNiPs have been tested,
and you're associating them versus dogs
that have the disease and don't have the disease,
and is there a difference,
and then on the Y-axis here is the P value.
And so what you can see is that
you know, you're not really doing anything,
there's not much of a difference,
and then suddenly on chromosome 37, it's like whoa,
so there's is something going on at chromosome 37,
and in this area formed what we call volcano.
So, essentially, there were SNiPs that sort of came up
and then finally one that's like hello,
and that's tagging the disease.
So another example of this was squamous cell carcinoma
of the digit, so skin cancer on the toes,
and it happens in a number of dogs,
and this study, they ended up actually finding
mutation that causes this,
and again, if you look at this,
there's 31 cases and 34 controls.
It's very few, few dogs,
but in this case they used pure-bred standard poodles,
scanned the genome using GWAS and ended up
finding an area on chromosome 15 of interest,
and went further on, and actually did find the mutation.
Now what about complex diseases.
So, this gets a little bit trickier.
So, one of the, to me, most outstanding papers
on complex disease that's come out
came out in 2014 on schizophrenia.
And what they found was 108 associated loci,
so when we talk about there's a lot of different
genes that contribute to this,
this is what we're sort of discussing.
So, this line here is their line of significance.
What's particularly outstanding about this
is that in order to do this study,
they had however many million SNiP markers
and over 150 thousand people in this study.
When you think about how much data, energy,
time and money it takes to do that,
it is absolutely outstanding.
So, if you have a dog model,
you might be in a little bit better situation.
So, the key point here is that dogs
really have a unique genomic architecture,
and linkage disequilibrium in dogs, like we discussed,
is a lot greater than it is in people, so again,
you get smaller sample sizes, you have few markers,
and we know that GWAS in dogs can be used successfully
to identify regions of the genome
that are associated with disease traits,
and obviously it's a big bonus,
if you ever find a disease marker in a dog,
that relates to a person,
you now have a one health situation.
You've really benefit a lot of species.
Okay, this is my, take a drink time.
So, getting back to laryngeal paralysis.
It has a very strong breed predisposition,
which really suggests a genetic basis.
In veterinary medicine, it's very common
that there are certain breeds that get certain diseases,
to the point where, if you know the signalment,
in other words, if you know the age, the gender,
the neuter status, and the breed of dog,
and someone tells you what the clinical presentation
problem is, you're going to have a pretty good idea
of what's probably going on.
I mean, there's always unicorns,
but there's a lot of horses.
So, when you've got a breed that is very commonly
gets a disease, and no other dog breed really gets it,
you have to be thinking to yourself,
you know it seems like it's probably something genetic.
And we look at pedigrees of dogs in this study,
it really does indicate that laryngeal paralysis
is heritable in the Labrador.
So, we've done a GWAS and right now,
we've got 81 Labrador Retrievers.
So we have 60 cases, and 21 controls.
And you'll understand why we have more cases than controls
in a moment, but the cases were all seen
at UW Veterinary Care.
They received either surgical treatment
for laryngeal paralysis,
so they had that tie-back surgery,
or they had clinical signs
that were very classic of the disease.
Then we had 21 controls.
So these dogs are all--
I think they're actually 12,
but 11 and a half to 12 years of age.
They've been checked with us at least twice.
They have no signs of respiratory obstruction.
They have no signs of progression of neurologic disease.
So, we're pretty confident
that they won't be getting the disease.
And all these dogs are screened for relatedness.
So, we get pedigrees from them,
and if there's any direct siblings we exclude them.
So, just to throw a Q-Q plot at you.
Essentially, this is one of the ways of looking
at our results.
So, what this is is an observed versus expected P values.
The basics of this is, if you look at
this line right here.
I'm going to use my mouse.
This is what--
And each one of these little dots are SNiPs.
Alright, so these are all what we kind of expect the SNiPs
to be, and then, suddenly at the top,
there's a view that break away because
what we're observing is not what we expect,
because they're associated with the disease,
because they're different.
And these SNiPs all lining up along this line like this
tells us that we have corrected, statistically,
for relatedness of the dogs within the study.
So, I'm just giving you a little bit
of our Manhattan plot.
This is just one chromosome.
But what you can see here
is that we've created this sort of,
there's actually, almost two volcanoes,
but this is the one of interest.
So there's sort of these volcanoes of SNiPs coming up,
and there's one that's really significant
and this lies in an area of the genome
that there's a number of genes that are
pretty interesting, that could be associated
with neurologic disease.
Perhaps more interestingly, is when you look at
our phenotype-genotype table.
So along this side, these are cases of dogs
that have the disease, and these are controls,
so like I said, we have 60 cases,
we have 21 controls, we have a total of 81 dogs,
and now we get back to our brown-eyed, blue-eyed.
So, you can have homozygous major,
in other words your big B big B.
All of our cases, or all dogs that have this major allele
have the disease, and then you have heterozygous dogs,
so big B little b, you know, you brown-eyed heterozygote,
and in these, you got 28 cases, and you have 4 controls.
And then little b little be we'll get to in a minute.
But what this map essentially, really strongly indicates,
is that this is a dominant disease.
So, just like with brown eyes,
if you have one of these big A alleles,
there's a pretty good chance,
you're going to get the disease.
Now these homozygous minor alleles don't totally match up,
because there are a number of dogs that get the disease
that you wouldn't expect to based on their genotype.
And there's a lot of reasons this can happen
with GWAS, for instance,
this SNiP is probably is probably tagging an area
of the genome, but it's probably not the actual mutation
that's causing the problem itself.
This could be more than one disease.
And so just like in CMT, in Charcot-Marie-Tooth,
where there's a lot of diseases
with a common clinical picture,
it's probably true in dogs too,
that there's a lot of diseases that could have
a common clinical picture.
My hope is that it's not true within a given breed,
but it's certainly a possibility.
You could also have, modifier locus,
so in other words, a dog maybe needs to have this,
or this contributes to part of a disease
but there's actually something else happening
nearby that is either supporting it,
or preventing the disease from happening.
So, there's a lot of reasons why
you could end up with dogs in this situation.
The other issue is that our phenotyping
might not be perfect.
It can be difficult, especially earlier on in this disease,
to be absolutely sure a dog has a condition,
which is why we recheck these dogs fairly frequently.
So, what about the whole Labrador population.
So, fortunately, because we do a number of genetic
studies on Labradors, we actually have a whole set
of dogs' DNA that has gone through these SNiP chips,
that are not associated with this study.
And a lot of these dogs admittedly
are from the Madison and Wisconsin area,
but they are actually nationwide.
So, we have about 210 additional dogs
that aren't enrolled in the study,
that were not recruited for anything to do
with laryngeal paralysis, that are pure-bred Labradors,
and if you look at their risk allele
in this situation, you have the very unfortunate realization
that this disease could be affecting
even up to 70 percent of older Labradors.
Now, clinically, when you go into a hospital
and you see a lot of old Labs, that's not surprising.
There are a lot of dogs that get this disease.
So, the key point is that we've found a region in GWAS.
It has number of candidate genes,
so we're really excited about that.
It does appear that this is an autosomal dominant trait,
although we do need to do more statistical analysis
on our pedigrees to really confirm that.
And, at least in the dogs that we have in our laboratory,
you know, up to 70 percent of the population
may be at risk of developing this disease in old age.
So, what's next?
Well, we need to look at this region a little bit more,
and so, we've just started an experiment
looking at whole genome sequencing,
which is really exciting,
with the hope of eventually, not only identifying mutation
but potentially even being able to create
some sort of a genetic test for this.
We also need to evaluate this in other breeds,
because like I mentioned,
could this be a clinical phenotype
where you have laryngeal paralysis
that's actually a result of a number of different
genotypes, where there's more than one disease
causing this problem.
So overall, I hope that I've at least introduced
you the idea of the comparative value
between dogs and people with one health medicine,
where dogs are really very valuable models for us.
The huge benefit from a veterinary perspective
is obviously, if you find a disease in dogs,
and it applies to people, you know you've got,
a strong funding source to continue to look at this
in dogs and you have a mutual benefit for both species.
I do need to disclose my funding.
And, you know, it takes a village to raise me.
So, I'd like to thank all of my collaborators and mentors,
but particularly all the owners and participants
which come from throughout the United States and Canada,
and mail in their DNA and help us out
beyond what I can describe to you.
I'd like to thank you,
and I can take any questions you might have.
[audience applauds]