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Where Basic Science Meets Public Health
One of the approaches Buxbaum uses to study this disease
is epidemiology. He is genotyping the TTR genes in African
American participants in two large studies of cardiovascular
risk in the African American community.
In these studies, the patients are genotyped using DNA taken
from blood samples and they are followed over the course of
many years to see whether they develop heart disease, and
if so what type. The study also collects and tracks relevant
data, such as electrocardiograms, echocardiograms, and chest
x-rays, which can all be used to monitor the functional state
of the heart.
The goal of the studies is to relate the presence of the
mutation to the occurrence of heart disease and age of onset,
as well as to explore possible interactions of amyloidosis
with other heart conditions. If the mutation-associated risk
for heart disease can be accurately determined, then a simplified
genotyping might become a useful diagnostic test, and genotype-positive
individuals can be given inhibitory drugs when they become
"We already have some data that suggests the allele we are
looking at is associated with an increase in mortality," says
Buxbaum. "If you look at the prevalence of the allele with
age, it goes downthe older [the population,] the fewer
people who have it.
There are also more basic questions that Buxbaum is interested
in addressing. "What is it that keeps this from happening
until late in life?" he asks. "And why these tissues only?"
Somehow the body keeps the heart free from fibrils throughout
decades of normal operation. Since the defects in the TTR
gene that cause the disease are genetic, the abnormal protein
is present in the circulation throughout life, yet doesn't
deposit until after age 60.
One possible explanation is that there are mechanisms that
take care of the misfolded proteins in the bloodstream, but
these mechanisms decline late in life. TTR spontaneously forms
misfolded fibrils in vitro in a short amount of timedays
or weeks. This does not happen nearly as quickly in vivo,
which is good evidence for some as-yet-undetermined misfolding
Another possibility is that the oxidation of TTR proteins
enhances the misfolding, a theory that has as its premise
the known fact that oxidative damage increases with age. If
TTR oxidation is linked to cardiac amyloidosis, then one's
chances of developing those complications would increase with
Yet another possibility is that the binding of TTR to other
molecules changes with age. It is possible that the affinity
for TTR of some proteins in the affected tissues increase
with age while the TTR binding of molecules that keep it soluble
in the circulation decreases.
Alongside these basic questions of the mechanism of cardiac
amyloidosis, there is, of course, the question of what to
do about it.
Possible Therapies and a Powerful Model
Recently, Professor Jeffery W. Kelly and his colleagues
in the Department of Chemistry and The Skaggs Institute for
Chemical Biology discovered a novel technique for dealing
with TTR fibrils in another, unrelated amyloid disease. Their
strategy is to introduce another protein that interacts with
the mutant protein and prevents misfolding by preventing dissociation.
A "suppressor" TTR subunit incorporated into a TTR tetramer
with disease-associated destabilizing subunits prevents the
tetramer from dissociating into potential fibril-forming monomers.
Significantly, they found that incorporating even one of the
suppressor subunits into a tetramer where the remainder of
the subunits have disease-associated mutations doubles its
This "trans" suppression approach may form the basis for
a new therapy for various blood-borne amyloidoses in which
the patient would receive an injection of the suppressor protein.
"I'm very excited about pursuing these potential therapeutic
opportunities," says Kelly.
Another, more traditional means of treatment involves using
inhibitors that block the binding of the misfolded monomer
to itself. Kelly and his colleagues have discovered a series
of small molecules that inhibit fibril formation in vivo.
Using these or similar inhibitors may become a useful strategy
for treating amyloid diseases.
Inhibition studies are important, says Buxbaum, "because
what you would really like to do is to prevent this genetic
Buxbaum says he came to TSRI in order to strengthen his
working relationship with Kelly and increase the rate of progress
towards an effective treatment. In order to address this,
the Buxbaum laboratory has developed a model for observing
the progress of the disease in vivo. Using this model, he
can test the efficacy of possible fibril-blocking therapeutics.
This provides more insight than simply looking in vitro,
since the physiological changes that lead to cardiac amyloidosis
take place in the context of many other proteins and signals
in the bloodstream.
The transgenics have the human TTR gene inserted into its
genome, and a certain percentage develop fibrils with an age-related
delay, analogous to that in humans. Furthermore, less structured
deposits that appear to be precursors to the amyloid fibrils
develop in both the heart and kidney in a larger percentage
Using this model, Buxbaum and his colleagues can also study
the basic process of deposition, observing the buildup of
the fibrils in living tissue over time. And they can relate
the molecular changes in TTR to what is happening in the heart
that may be responsible for the change in behavior of the
protein with age.
"We're just beginning to look at [these questions] in our
model," says Buxbaum. "The sequential events that happen from
the time the protein is synthesizedwhere it goes and
what it does."
Insight into ths in vivo process and how it can be
slowed or stopped using the molecules found to work in the
test tube is one of the two ultimate goals of the Buxbaum
laboratory. Helping to make these molecules available to individuals
found to carry the responsible gene before they get sick is
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