Key to Triplet Repeat Brain Diseases Open Door For New Way To Understand, Treat Genetic Diseases"

April 29, 1996

DURHAM, N.C. -- As reported in the March issue of Nature Medicine, Duke University Medical Center researchers have found a possible key to brain disorders such as Huntington's disease, as well as discovering a new concept of how mutant genes may produce disease.

The scientists discovered that these "triplet repeat" disease genes produce proteins that errantly stick to an enzyme crucial to the production of energy in brain cells. They believe that because this enzyme cannot, therefore, do its job, brain tissue will malfunction and may, over years, die. This would result in fatal physical and intellectual deterioration, such as that seen in Huntington's disease patients.

Furthermore, their findings may answer why the number of genetic repeats in these diseases determines how severe symptoms will be: More repeats produce a protein that holds this "energy" enzyme more tightly, producing widespread nerve cell failure in the brain.

The work offers biomedical researchers a new way to understand, and to potentially treat these genetic diseases, the Duke scientists say.

"This is the first explanation of what these proteins produced by triplet repeat genes may be doing in nerve cells," said neurologist and principal investigator Dr. Warren Strittmatter. "It provides a new paradigm for inherited neurologic disease, and that is the repeat region of these proteins binds other proteins critical to glucose and oxygen metabolism in the brain. And the larger the repeat region, the tighter they bind."

Authors of the study are from the same team that discovered the major gene linked to Alzheimer's disease. In many ways, "this finding may be more pivotal than our earlier work," said Dr. Allen Roses, scientific director of the Deane Laboratory at Duke, where the experiments were conducted. "We are beginning to cross the difficult area between gene discovery and abnormal functions of the brain, and to open the door to target drugs," he said.

The primary researchers involved in the discovery are Duke neurologists Strittmatter, James Burke and Jeffery Vance. Other collaborators include Jan Enghild and Margaret Martin, both from Duke, and geneticists Yuh-Shan Jou and Richard Myers from Stanford University School of Medicine.

Abnormal "triplet repeat" genes constitute the molecular equivalent of stuttering. They repeat the same sequence of genetic units, called nucleotides, over and over far more than do normal genes. While a normal gene might repeat the three nucleotides cytosine, adenine and guanine (CAG) fewer than 30 times, genes that produce Huntington's disease and other such brain disorders might repeat the sequence between 40 and 100 times. As the repetition grows, the disease becomes more severe.

The Duke study explains why the number of nucleotide repeats in such diseases relates to the severity of symptoms, the researchers say. The nucleotide sequence CAG determines that the amino acid glutamine will be inserted into the protein during its translation from genetic information to protein in the brain cell. The greater the number of CAG repeats in the gene, the more glutamines will be added, resulting in a structurally different protein that binds strongly to a particular energy enzyme. As these crucial enzymes become disabled, increased numbers of neurons malfunction and may die, destroying brain tissue.

The researchers have found this effect in two neurological diseases -- Huntington's and Haw River Syndrome. They believe the effect also works for at least three other rare neurological disorders -- Kennedy's syndrome, Machado-Joseph disease, and Spinocerebellar atrophy I -- that have the same expanded CAG repeat.

"Traditional dogma is that mutant genes produce proteins that either don't work at all or don't work well," said Burke, the first author. "However, we found the proteins produced by these genes interact in a different way, based on the number of repeated amino acids, producing devastating consequences."

"The novel insight is that this extra domain produced by triple repeats acts in the same way across all these diseases," Vance said. "Even though we don't know what the normal function is of each of the disease genes, the triple repeats are causing all the proteins to bind to this single enzyme. Scientists have usually only thought about how a gene mutation produces a change in that protein that results in a disease. This is an observation that provides a mechanism for a group of diseases."

If drugs could block the protein interaction the researchers described, then the discovery may offer treatment options, according to Roses.

The investigators became interested in the action of repeated gene sequences in brain tissue two years ago, when they discovered Haw River Syndrome (also known as DRPLA), an inherited neurological disorder that affects residents of a rural North Carolina community.

The scientists found that the syndrome was like Huntington's disease in that it was attributed to genes that acquire excess CAG nucleotide sequences.

Although the scientists do not know what the roles of the proteins produced by Haw River or any of the triplet repeat diseases are, "Our hypothesis is that these different proteins are all doing something similar, which is binding to the same protein," Vance said. In other words, the abnormal repeat in the DNA is adding a "docking area" domain in the protein, which allowed substances that bind loosely to the normal protein to stick more tightly to the enlarged disease protein.

To test that theory, the Duke team made artificial strings of glutamines, either 20 glutamines, found in normal proteins, or 60 glutamines, found in the mutant proteins. They then mixed these glutamine strings with brain proteins to see what proteins bound. They found that one brain protein, glyceraldehyde-3 phosphate dehydrogenase (GAPDH), bound to the long glutamine string better than to the short glutamine string. GAPDH, found in all cells, is known to produce energy, along with a number of other important functions.

In a second experiment, brain proteins were incubated with GAPDH. Huntingtin protein and DRPLA protein bound to GAPDH, while other brain proteins did not.

While he can hypothesize that binding of GAPDH may start the pathway of errant protein interactions, Strittmatter said he can't yet define the cascade of events that produces the disorders. "The brain is entirely dependent on glucose metabolism, and this is a protein that is important in that pathway," he said. "But because GAPDH is expressed in every cell, not just in brain cells, there has to be another level of complexity that we don't understand."

"This is at least an initial insight into the molecular genesis of these diseases," Burke said. "It is a novel interaction that may explain the increase in severity with increasing repeat size."

The researchers point out that their theory of triplet repeat diseases would not apply to those that contain stuttering sequences in the "junk" area of the genome, since those areas do not encode for protein production. The genetic disease called myotonic dystrophy is an example of such a disorder.

The interdisciplinary research under Roses was a key to the discovery, Vance said. "We are a group of clinicians, geneticists and molecular biologists. Only by working across disciplines could we have come up with such an understanding," he said.

Additional funding or support for the study came from the Joseph and Kathleen Bryan Alzheimer's Disease Research Center at Duke, the Duke Center for Human Genetics, and the National Institutes of Health (specifically the National Institute on Aging, and the National Institute of Neurological Disorders and Stroke).

Duke University

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