Science Editor

Two young members of an extended French family recently succumbed to congenital heart failure. One, a 37-year-old man, was running up a hill when he suddenly dropped dead of exertion. The other, an elementary school child, slumped lifeless to the floor when startled by being called on in class.

This numerous pedigree goes back four generations for a total of 88 men, women and children, of whom 23 are affected by this potentially fatal cardiac disorder, according to genetic analysis. "Theirs is a dominant disorder," observed cell biologist Vann Bennett at Duke University in Durham, N.C. "That means half of their children will have it."

Bennett is senior author of an article in Nature dated Feb. 6, 2003. Its title: "Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death."

"We found this human family in France," Bennett told BioWorld Today, "which has a mutation in a protein that was quite unexpected - so ended up in Nature. At this time, it's the first example, so we'll have to see how common this particular disorder is. How it got to us at Duke," he continued, "is a sort of scientific detective story."

Bennett recounted: "I had actually discovered the first ankyrin protein in 1978 in human red cells, and my co-authors and I subsequently found that ankyrins are present in most cells of the human body. We used knockout techniques to make mutant mice that were lacking in ankyrin genes. These KO animals, minus the ankyrin-B protein, had abnormal calcium dynamics in their heart cells. We also found that the mice themselves displayed EKG abnormalities that were very similar to those reported for this French family.

"So then I contacted the researchers in France," Bennett went on, "and suggested that ankyrin-B might be the gene mutated in the family. The scientists had reported that there was such a family, and they mapped the gene to the long arm of chromosome 4, in a paper published in 1995. That's when I knew about the family.

"They mapped the gene, and we were working on the mouse end of this ankyrin-B protein, and came up with a mouse that closely matched the family's genotype."

Bennett noted, "A problem you run into when doing genetic analysis is, What if you encounter a mutation that's functionally silent? How did we know that this mutation the French found causes the disease? Was it merely a polymorphism? One test we were able to provide was we had cardiac cells from our mutant mice. We added back the human gene to these cells and they became normal - beat normally and had normal calcium waves."

True Mutation, Not Mere Polymorphism

"But when we did that with the mutant ankyrin that they had discovered in the families, the cells did not recover, did not become normal. This was evidence that the mutation they found in the family's DNA was actually a functional mutation, not a polymorphism. End of detective story.

"Before launching our mouse experiment," Bennett related, "first we had to find the other members of the ankyrin gene family. So we used molecular biology to clone the genes encoding all three protein subtypes: ankyrin-R for red cells; ankyrin-B for brain, where we originally purified it; and ankyrin-G , standing for giant,' a very large protein. Then the next step was to look at their function. So we knocked these genes out in mice. With the ankyrin-B KO mouse, we originally noticed that in addition to major nervous system problems in homozygotes - when both copies of the gene were removed - these mice died at birth. And they had major problems in their nervous systems, which we expected. But they also had muscle problems - an elevated enzyme that's released from muscle that's damaged. And we also noticed some other problems in their skeletal muscle.

"We found this protein very abundant in cardiac tissues, so we grew heart cells from these mice and noticed that they didn't beat normally. A heart cell in culture will actually beat - contract. These cells did not beat normally. Then, based on the biochemistry, when we knew that the ankyrin can bind to several proteins that are important for calcium homeostasis, we evaluated these proteins in both the adult and neonatal cardiomyocytes. They were reduced, and also altered in their localization. And the patterns of calcium release were abnormal.

"Meanwhile, the French investigators had described a syndrome in their families, which they called Type 4 long-QT cardiac arrhythmia.' Long QT refers to changes in the electrocardiogram, which has components that allow a cardiologist to tell how regular the cardiac rate is, and how well the heart recovers after each contraction cycle. The QT interval, when prolonged, means the heart has trouble repolarizing - that is, recovering from a beat. And during that slow-recovery phase, the patients are susceptible to a second contraction cycle, which can lead to chaotic beating and loss of cardiac output. That's the cardiologists' name for the French family. They call it long-QT type 4. So this led to conjecture that the ankyrin gene could be the basis for the long-QT-type 4 syndrome."

Bennett observed, "Ankyrin is ubiquitous and a Mesozoic invention. Plants don't have it, nor fungi nor bacteria - just animal cells. In the heart, the readout is muscle contraction. So far, based on our mouse work, we have evidence for other disorders or diseases beyond the heart. There's another ankyrin important in vision, cognitive function and beta cells. Beta cells in the pancreas are also excitable membranes, and they respond to glucose and secrete insulin, broadening the concept of excitable membranes."

Fine-Tuning Ion Channels

"Normally, ankyrin-B acts as a biochemical symphony conductor, ensuring that microscopic pores in heart muscle cells - known as ion channels - open and close in a coordinated fashion. These channels allow such chemicals as calcium, potassium, sodium and chloride to pass in and out of the cell with each heartbeat, thereby regulating the heart's electrical activity.

"So I think our new mechanism opens a door to a very large room," Bennett concluded, "with much to be learned in many organ systems."