Tip:
Highlight text to annotate it
X
Dan Kastner: And thank you for the invitation to speak
here today. It's really terrific. This is very, very exciting program and it's a privilege
to be speaking here. So in any case, I will be talking for the next few minutes about
some work that has been passion of my professional life for the last over 20 years, and that
has been understanding some of the disorders that present as undiagnosed hereditary fevers
in patients and over the course of time has had really unexpectedly, I think, large implications
for understanding of the regulation of implementation in man.
So in any case, let's get into it. And certainly in terms of our thinking about charting course
for genomic medicine, at lease for us, it's been very much a matter of starting with particular
patients with particular phenotypes, and so I will just regal you with a few of them over
the next of the next minute or two.
First of all, here is, at the time this image was taken, seven year old girl from California
who the months long episodes of fever with severe abdominal pain, migratory rash, and
sometimes arthritis. And you can see here there is, actually, laparoscopic view of her
abdominal cavity showing adhesions that had formed as a result of recurrent episodes of
inflammation. Here's a toddler from Baltimore who had a different type of recurrent fever
syndrome, also hereditary. And in this case this patient had week long episodes of fever
with diarrhea, the skin rash, and cervical lymphadenopathy. This is a kidney biopsy looked
at under polarizing light showing amyloid deposition in a young man from turkey who
came into United States as a graduate student at Georgetown and actually was seen in a screening
physical examination with pro anuria and turned out to have amyloid deposits in his kidneys
and incidentally had a history of a few day episodes of fever intermittently over the
course of the first 20 years of his life. And then here is an audiogram taken from a
young woman in her early 20 from Texas with high frequency hearing loss associated with
the syndrome of single day fevers. Just a little bit more, we have here an MRI of the
brain of a young child with chronic aseptic meningitis. This is the flair image and shows
basically the white is the inflammation on the meninges.
This patient has condition called NOMID, Neonatal Onset Multisystem Inflammatory Disease. This
is a young man from Kansas City, whom we take care of at the clinical center, who has a
syndrome in which he develops pyoderma gangrenosum. That's shown here. It's basically a breakdown
of the skin. These patients develop a purulent area on the skin and bleeding. This particular
lesion lasted for about a year and was refractory to high doses of corticosteroids and required
high doses of narcotics to control the pain. This is a child from Puerto Rico, a seven
year old, who had a new disorder that I'll tell you about in a few minutes. It's something
called DIRA, Deficiency of IL-1 Receptor Antagonist. And then, finally, the last image I'll show
you is of a autopsy specimen of the father of one of the patient that we follow at the
NIH with a different recurrent fever syndrome. A fever syndrome that presents with prolonged
fevers, periorbital edema, migratory rash, and abdominal pain. Unfortunately he lived
a little bit too early for the genome project and passed away before we started seeing his
daughter.
All of these patients have diseases that have basically been susceptible to the analysis
of the genome project, and basically we have an explanation for the illness in all eight
of these cases that I'm telling you about right now. And luckily, both for us and for
the patients, we actually have treatments targeted treatment for each of these patients,
except for the last of course. And essentially these targeted treatments are based on our
understanding of the path of physiology that came out of the genetic studies and the fortuitous
parallel universe of immunology and biotechnology that developed cytokine antagonist that effective
in treating these patients. So anyway let’s just talk a little bit about some of these
diseases in more detail.
First, we'll start with this patient here a young man, a young Armenian man, that I
saw at the beginning of my rheumatology fellowship here some 25 years ago. I was only five at
the time.
[laughter]
And in any case, he had recurrent episodes of fever and arthritis that would last a few
days at a time. He turned out to have Familial Mediterranean Fever, at least by clinical
diagnosis, which led us to get interested in the positional cloning of the gene for
this disease. Of course this was back, at the time, in the first phase, if you will,
of the genome project that Rick was referring to, and certainly the paradigm of cystic fibrosis
was siren song for us. And so we mapped the genes for this disease to chromosome 16, having
looked at every other chromosome before finally localizing the causative locust to chromosome
16. And then became the genome project for this area of the human genome, and eventually
narrowed the interval to 200,000 base pair interval. There were 10 genes that we sifted
out of this region, and of course , it is the 10th of the 10 that turned out to have
mutations associated with disease, hitting codes what was then a novel protein that we
called pyrin after pyrexia for fever. And a competing French group denoted Mare Nostrum,
they being a lit bit more erudite then we, after Mare Nostrum for the Latin after the
the Mediterranean sea.
In any case we'll talk a lit bit more about this protein in a bit. But just to turn to
another of these diseases this is back actually of the man whose forearm I showed you a few
minutes ago. You can see severe cystic acne in his case. And we were interested in this
phenotype and we were also interested, at the same time, in proteins that interact with
pyrin, the FMF protein. And by, basically, a comprehensive approach using these two hybrid
studies, we found that one of the proteins that interacted with pyrin is this one here
PSTPIP1, proline-serine-threonine phosphatase interacting protein. Basically, it's a pyrin
binding protein and mutations in it have an effect on the lividity [spelled phonetically]
of binding of this protein to pyrin and actually lead to increased IL1 secretion in peripheral
blood leucocytes from these patients. See we have both pyrin and a pyrin binding protein.
Here is another patient. A young man named Jonathan who was referred to us from North
Carolina who had a different syndrome. He actually had fevers, a hive like rash, and
evidence of central nervous system inflammation, and over growths of the epiphysis of his long
bone leading to this knobby knee appearance. In any case, he has mutations in not a pyrin
binding protein but a pyrin homolog. So there is a family of proteins, that we'll talk about
in a bit, that have an in terminal domain that's known as the pyrin domain, not the
marenostrin domain but the pyrin domain. And actually these proteins are involved in the
regulation of inflammation in patients who have the disease that he has, NOMID, Neonatal
Onset Multisystem Inflammatory Disease, have mutation, not in the pyrin domain but in an
aggregation domain in the middle of the protein. And actually there are other phenotypes that
are also associated with this as well.
So we have pyrin, a pyrin binding protein, a pyrin homolog, and now we'll turn to another
patient who was referred to us from Newfoundland, Canada, who has this pustular rash all over
his body. This is an image taken when he was nine months old. This is actually the fold
of the neck, so these are just pustules all over the back. And this patient has mutations
in the IL-1 receptor antagonist gene. So this is the gene actually in same path way, and
actually there are several other patients, but we have identified with other mutations
in this gene but the same phenotype leading to the new disease, deficiency in IL-1 receptor
antagonist. Here is a photo of a family that have yet another phenotype. This is phenotype
actually this girl's father is the one who's autopsy slides are featured issue of cell
that I showed you awhile back. She has long episodes of fever with serosal and joint inflammation.
In patients who have this, actually have mutation in another inflammatory molecule, another
molecule that's important in regulation of fever in humans and that is the p55 TNF receptor
or TNF R1 A. Patients who have this disease, many of them have mutations at cystine residues
that have involved in holding the folding structure of the cystine rich domains in place
in the extra cellular part of this protein.
This is yet another patient who has a disorder. She's from Chicago has disorder in which the
episodes of fever are shorter and they are caused this is work of a group from Netherlands
in mutations in mevalonate kinase which is an enzyme involved in cholesterol biosynthesis.
And then, finally, the last of these I will at least mention is the Behcet's Disease which
in counter distinction to the other disorders I told you about, is a disease with complex
inheritance, and Behcet's Disease is characterized by the triad that's illustrated here: oral
ulcerations; ocular inflammation this is hypopyon Uveitis. This is the accumulation
of pus in the anterior chamber of the eye and genital ulcerations. And so we have been
involved in a genome wide association study in this disorder and basically looking at
about 1,200 individuals with Behcet's Disease from Turkey in 1200 Turkish controls and then
conform firming it with cohorts from Japan, Korea, and number of other areas. We found
that actually there's a variant of the I-L10 locust that is highly associated with Behcet's
Disease in all of these populations with a modest risk but a biological story that goes
along with it. And so individuals who actually have two copies of the risk aliel [spelled
phonetically] produced less I-L10 than individuals who have either one copy or who are homozygous
with the non risk aliel. That's interesting because I-L10 is an anti inflammatory cytokine,
so it actually does give some account of how it is that people with Behcet's Disease might
develop that phenotype.
So in any case, that's just a little overview of the genetics, what we've learned in terms
of diagnosis. What have we learned in term of biology? Well, there's a lot more biology
than what I can probably talk to you about in the next two hours that I have for the
talk, so I'll try make it brief. But in any case, it turns out that pyrin itself, the
first protein that I told you about which is mutated in FMF, is the prototype for a
domain that's found at its interminus [spelled phonetically] which is a six alpha helix domain
that at least often times can form a dipole that is involved in homotypic interaction.
And so pyrin domains and interact with other pyrin domains. In case of pyrin itself, it
interacts with an adapter protein called AFC which in turn can interact caspase one, the
enzyme that involved in the activation of pro IL-1 beta to IL-1 beta itself. And actually
the pyrin domain is now known to be a motif that's found in a whole large category of
proteins that have involved in the regulation of inflammation in cell death. And this is
just a phylogenetic diagram that indicates some of the proteins that have pyrin domains
in them. Many of them are the NLRP family of proteins which are shown here with the
in terminal pyrin domain shown in green.
So in any case, if one then thinks about the biology of a disease like FMF, we've actually
knocked in the mutations and the FMF gene, at least some of them into mice. This is the
work of Jay Chay [spelled phonetically] in our lab. And you can see that in mice that
are knock ins for the V726A mutation you may not be able to see this, but these mice actually
spontaneous developed arthritis. And if one sections the joint, one sees the characteristic
influx of polymorphonuclear leukocytes into the joint as one would see in human FMF. And
if one breeds these mice on IL1 receptor knock out background one can see that actually there's
a marked reduction in the inflammatory phenotype that these animals have. The pyrin domain
protein, actually, at least one of them, and LRP3 is actually the an important mediator
in a macromolecular complex that's call the inflammisome that's involved in IL-1 beta
activation. And this slide just illustrates the IL1 activation pathway and the various
phenotypes that we and others have seen that are associated with molecular lesions in this
pathway. So that individuals who have NLRP3 mutations develop hive like rash and a spectrum
of other inflammatory phenotypes including CNS inflammation. Patient with FMF who have
mutations at another point in that pathway get a different type of rash and a different
duration of episodes. Patients with a hyper IGD syndrome have mutation in regulator of
that pathway. Patients with the deficiency of IL-1 receptor antagonist get yet a different
phenotype illustrated here with a defuse pustulosis. Patients with mutations in PSTPIP1 have the
pyoderma gangrenosum phenotype. And then harkening back to the first talk of the second part
of the the session here, this is actually high dock to defurum wall [spelled phonetically]
and recurrent high duct to defurum wall is actually an inflammatory condition which can
be associated with variance of NLRP7, one of the regulators of this pathway.
We've also learned a lot about other inflammatory disorders. In the case of the TNF receptor,
actually, we know now that misfolded protein, actually, can form aggregates in the cell
which are involved in assembling a signaling cascade which then lead excessive cytokine
production by leucocytes from these patients. Moreover, we've gotten a much better understanding
of various disorders of inflammation. And a few years ago we had proposed a categorization
of inflammatory or immunologically mediated diseases in which disorders with auto antibody
formation and antigen specific T cells we think of as auto immune diseases disorders
in which these antigen specific variants are not present auto antibody or antigen specific
T cells are not present we think of as auto inflammatory diseases. And this image just
remind us of the dichotomy between the adaptive immune system in which lymphocytes play a
role and in which the receptors somatically rearrange or mutate and the innate immune
system in which the cell types are, for the most part, myeloid in nature which the receptors
do not somatically rearrange or mutate. And so basically the auto inflammatory diseases
are diseases of innate immunity, and this table here just simply illustrates there are
now lots of diseases besides the periodic fever syndromes that, in fact, fall under
this auto inflammatory rubric [spelled phonetically]. And a couple of them that are much more common
diseases are: gout, in which uric acid crystals actually activates the inflammisome, and this
then does suggest, and in fact led to therapeutic trials IL-1 inhibitors in gout; and atherosclerosis.
This is a paper by Ikey Lot's [spelled phonetically] group from a year ago basically demonstrating
mouse models of atherosclerosis. But if one looks at knockouts for IL-1 pathway that the
we don't see atherosclerosis in those animal models. And so atherosclerosis is probably,
at least to some extent, auto inflammatory as well.
We've also learned a lot about human history by tracing spread of these mutations across
different populations. And so here is I've just shown the spread of various FMF mutations
in the Mediterranean Basin. And, in fact, there's an extraordinarily high frequency
of these mutations in these populations different mutations in different populations. Mutations
cluster around what looks to be a binding pocket in the C terminal domain of these proteins
suggesting that there probably is some sort of a selective pressure, although we don't
know yet what it is. In the case of the deficiency of IL-1 receptor antagonist we know that there
are certain areas Newfoundland, the Bible belt the Bible belt of the Netherlands, and
northwestern Puerto Rico where we sometimes see founder effects. And particularly in northwestern
Puerto Rico, there actually have been additional patients identified from the original propositus
patients. And then finally for Behcet's Disease, many of you know Behcet's Disease is distributed
along the Silk Route of Marco Polo going from Venice through the Middle East, Korea, and
Japan. And in point effect the variants that we've seen both IL-10 and the IL-23 receptor,
which I didn't talk about, they are seeing both in the Turkish population and far Eastern
population, suggesting that more than silk was traded along the Silk Route of Marco Polo.
So in any case, then to get to I'm sure many are interested in as sort of the final vindication
of this kind of work, we have been we and our patients have been extraordinarily lucky
in terms of treatment possibilities. And as I said at the outset, this is at least in
part due to the fact there has been this parallel universe of immunology and biotechnology research
going on at the same time. So in the case of patient with NOMID, the disease that is
caused by mutation in NLRP3, the inflammisome protein, about four years ago Rafealla Obocmanski
[spelled phonetically] in NIAM set up a protocol to look at 18 patients with this disease with
anikenra [spelled phonetically] the IL-1 receptor antagonist which is basically an agent that
binds to type one IL-1 receptor but not to the accessory protein and thereby blocks IL-1
signally. And one can see that there is just an absolutely whoops dramatic response to
treatment. This is within three days of treatment these patient's skin rash goes away. Their
ocular inflammation goes away. Within about three months the CNS inflammation, the chronic
aseptic meningitis is largely gone. The arrow points the copelia [spelled phonetically]
this is coplear inflammation which dissipates as well with specific cytokine therapy.
Here is the patient with DIRA, the Deficiency of IL1 Receptor Antagonist, pustular lesions
all over the body at age 9 months. Within three days of beginning treatment with IL1
inhibitor you see the skin beginning to swaft [spelled phonetically], and the child is beginning
to smile rather than cry. And by seven days after the initiation of treatment you can
see that nearly all of the skin has swaft. And basically this patient is well as long
as he continues on the anikenra [spelled phonetically] injections.
Here is the patient young man from Baghdad, Iraq, who has Familial Mediterranean Fever.
The usual treatment for that is coltrazene [spelled phonetically], but he has severe
amyloidosis including in his gastrointestinal track with leads to chronic diarrhea, so he
was not able to tolerate coltrazene, and so instead we have put him on anikenra as well.
At the time that we first saw him his injection fraction was about 37 percent and had he had
chronic malabsorption and we really didn't think that he was going to survive for very
long at all. But, actually, on anikenra, he has done extremely well. His aniloid deposits
have at least to some extent regressed. And five years later, here's his picture after
having just eaten pizza for lunch.
And then finally thinking about some of the more common diseases that now have what appears
to be an auto inflammatory component, type two diabetes, so you might not think of diabetes,
especially type two diabetes, as having an inflammatory component. But in fact, hyperglycemia
induces islet cells to produce IL-1. IL-1 in turn is toxic to islet cells, so that in
fact they end up producing something that causes them to commit suicide which of course
leads to more hyperglycemia and more IL-1 production. And one can see that if one treats
these patients with an IL-1 inhibitor, then in fact glycemic control improves. And IL-1
inhibitors are actually now in clinical trials for type two diabetes.
Finally, I would just end with yet another disease that I haven't talked about yet. PFAPA
the syndrome with periodic fever with aphthous stomatitis, pharyngitis, and cervical adenopathy.
It's actually the most common recurrent fever syndrome in kids and we see it quite frequently
in our clinic. It is caused by complex set of gene that we don't yet understand. But
taking a gemonic approach to it, and just looking at gene expression, we found that,
in fact, patients with PFAPA during their attack have activation of the inflammisome
pathway, the IL-1 pathway. So we have started treating a few of these patients with anikenra
and we do see a diminution of in fevers and other episodes. Just earlier this week on
Tuesday I saw one of the patient that's in this study from Tennessee who actually has
taken now 20 courses of anikenra and has aborted all of his attacks, basically, within hours.
A little bit later that morning we saw a three year old from Seattle whose mother had lost
her job because she was having to stay home with her daughter with her attacks and fevers,
and, of course, we've now started the daughter on anikenra as well.
There's a lot left to be done. This is a pie chart of some 1,300 patients that we've seen
at the NIH with various recurrent fever syndromes, and you can see we only have genetic diagnosis
and about 38 percent of them, and so there's lot left to do.
So in any case, I'll just close with some acknowledgements to people that made a big
difference in terms of this work. Evonot Sintiavich [spelled phonetically], who's been the person
whose don a lot of the gene discovery work and terms of sequencing in our lab; Rafealla
Obocmanski, now a tenure track investigator in NIAM whose done a lot of clinical studies;
Jay Chay, whose developed the animal model; Seth Masters, whose done a lot of the work
on functional studies; Richard Seagull [spelled phonetically], a collaborator who does a lot
of work on cell biology of traps; and Elaine Remers [spelled phonetically], whose headed
up our GWAS studies; and then our larger group of the lab and clinical people shown at a
recent NHGRI retreat. And finally, of course, acknowledgment to the NIH clinical center
where all this work took place. So anyway, thank a lot.
[applause]