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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 available.

"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 down—the 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 time—days or weeks. This does not happen nearly as quickly in vivo, which is good evidence for some as-yet-undetermined misfolding correction mechanism.

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 age.

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 stability.

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 disease."

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 of cases.

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 synthesized—where 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 the other.


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Amyloid deposits in heart muscle caused by a mutation in the transthyretin gene, such as the ones apparent in this micrograph, cause progressive cardiomyopathy.





















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