Scientists have scored a major advance in understanding why muscles disintegrate in patients who have the crippling genetic disease known as Duchenne's muscular dystrophy -- a significant step toward improved diagnosis and, perhaps one day, an effective treatment.
In late December, researchers at Harvard Medical School and The Children's Hospital of Boston announced in the British journal Nature that they had discovered the muscle protein, which is missing or defective in Duchenne's patients. A little more than a year ago, the same team quickened the pace of muscular dystrophy research when they announced the discovery of the Duchenne's gene, whose absence or alteration causes Duchenne's. It is the normal Duchenne's gene that produces this key muscle protein, named dystrophin by the researchers.
With the discovery of the Duchenne's protein, medical scientists have their first hard clue to what exactly happens within muscle cells to cause the gradual wasting away of muscular dystrophy patients. Once the role of dystrophin in the normal muscle cell is understood, it might be possible to design treatments that could replace the function of this absent or defective protein in patients and stem the course of the disease.
"The discovery of this protein is the single greatest discovery on the path toward treatment that has ever been made. Period. Exclamation point. We are very excited about it," said Dr. Donald S. Wood, director of research for the Muscular Dystrophy Association in New York City. "We know for the first time what starts the disease in muscle."
The discovery of the Duchenne's gene and the dystrophin protein by Dr. Louis M. Kunkel, leader of the Boston research group, is one of the first triumphs of genetic engineering techniques, which are now being applied to a long list of inherited diseases. Laboratory methods developed over the last few years allow scientists to manipulate deoxyribonucleic acid (DNA), the chemical carrier of inherited information, in such a way that the specific DNA segments representing individual genes can be identified, reproduced in massive amounts and then used to manufacture the proteins that are produced by that portion of the genetic blueprint.
Once the defective Duchenne gene was identified last year, finding the protein made by the normal Duchenne gene was only a matter of time and painstaking, but predictable, lab work. The Boston research team showed that most individuals carry the normal Duchenne gene and make normal dystrophin protein in their muscle cells. But when the dystrophin-producing gene is absent or in some way defective, the individual fails to make normal dystrophin and develops Duchenne's muscular dystrophy.
"This is one of the first proteins identified in which its absence causes disease," said Dr. Kevin Campbell of the University of Iowa College of Medicine in Iowa City and one of Kunkel's collaborators on the dystrophin announcement. "This is a new area."
The excitement in the Duchenne's field undoubtedly will be played out again over the next decade as scientists identify the genes and their proteins that are the underlying causes for many of the more than 3,000 genetic diseases that have now been identified.
An intense race currently is under way to identify the cystic fibrosis gene, for example; a report earlier this year that the CF gene had been found proved incorrect, said Dr. Victor McKusick, professor of medical genetics at Johns Hopkins University and author of "Mendelian Inheritance in Man," a book as well as a recently computerized compendium of all known human genes and their associated diseases. But the researchers are close, and they are expected to find the culprit gene that causes cystic fibrosis within a year.
Similar efforts are under way for other genetic diseases, but none are as far along as the research on Duchenne's.
At the same time, scientists are cautious about raising public expectations. Even after the role of dystrophin has been well documented, it could take years before a new therapy becomes available.
Ultimately, the most effective treatment is likely to be the highly experimental technique of gene replacement therapy. Because muscular dystrophy, like many other genetic defects, is caused by the deletion or alteration of a single gene, Kunkel, Wood and other scientists predict that some day in the future, the disorder may be cured by replacing the absent or defective Duchenne's gene with a normal one.
The technique is now under intense development for a number of diseases. A group led by Dr. W. French Anderson at the National Heart, Lung and Blood Institute, among others, currently is developing a gene therapy technique for children born with severe combined immune deficiency syndrome -- the boy-in-the-bubble disease. In this treatment, bone marrow cells are removed from the patient. Scientists insert a repair gene into the cells and then put the treated cells back in the body.
A number of genetic blood disorders, such as the thalessemias or anemias, may one day be treated with this approach, McKusick said. It will be easier, he said, to get repair genes into blood cells than into muscle cells for muscular dystrophy or into the brain for disorders such as manic depression or Alzheimer's disease.
While in theory, human gene therapy may one day be used to treat muscular dystrophy, Kunkel cautioned that "gene therapy still has a long way to go."
At this point, scientists are focusing on a tantalizing clue in the new research that could lead to an effective treatment: The key protein dystrophin also is missing in a strain of mice, the mdx mouse, that has many of the signs and symptoms of Duchenne's muscular dystrophy, including muscle weakness. But most important, the animals do not become incapacited or die prematurely.
If researchers could understand how the mouse muscle compensates for the lack of dystrophin, then they may be able to lay the groundwork for new types of treatment for human patients.
The most immediate impact of the dystrophin discovery may be to improve diagnosis. Existing genetic tests only can identify about half of those with Duchenne's. Doctors must rely on clinical signs to distinguish Duchenne's from Becker's (or benign juvenile) muscular dystrophy. Becker's begins later in life than Duchenne's and tends to be more mild. Although patients may be wheelchair-bound as adults, they usually live into their thirties and forties and some into their sixties.
By testing a muscle sample for the presence or absence of dystrophin, physicians should be able to determine which disease is present, said Iowa's Campbell. Even though neither form can be stopped, a proper diagnosis could help patients and their families plan for the future and cope better with the physical and pyschological aspects of the disease as it progresses.With Duchenne's muscular dystrophy, the symptoms -- muscle weakness, waddling gait, frequent falls and difficulty standing up -- can become apparent in boys only 18 months of age. Because they have difficulty raising their knees, they may never learn, or be able, to run.
By age 12, most DMD patients are confined to wheelchairs. By 20, most are dead. The muscles of the diaphram deteriorate so much that the lungs fail, ultimately causing suffocation.
Although treatments to block growth hormone have been tried to slow the muscle deterioration, nothing delays or stops the progression of the disease among the 20,000 affected American boys. Most therapies merely ease the discomfort.
Duchenne's, the most common and the most severe of the several forms of muscular dystrophy, strikes an estimated one of every 3,500 boys who are born in the United States. It seldom affects girls because the Duchenne's gene is carried on the X chromosome, one of two sex-determining chromosomes. Girls inherit two X chromosomes, so if the gene is defective on one of them, it's probably normal on the other X chromosome.
Boys, however, inherit one X chromosome and one Y chromosome, each carrying a different set of genes. If the Duchenne's gene is defective on the one X chromosome inherited by the boy, he does not have another X chromosome to carry a normal gene and compensate for the defect. Consequently, the boy develops muscular dystrophy. Hemophilia, another genetic disease that only affects males, is inherited in the same way.
It's clear that once the defective gene is inherited, the body fails to produce normal dystrophin, which is why the muscles completely deteriorate over the next two decades.
To find the gene, Kunkel worked with material from a Duchenne's patient who was literally missing a piece of his X chromosome. Using a genetic engineering technique called subtraction hybridization, Kunkel matched up chunks of DNA from a normal X chromosome with chunks of DNA from the patient's abnormal X chromosome. Once everything was matched, the DNA left over represented genes in the normal X chromosome that were missing in the Duchenne's patient, including the gene for dystrophin.
Kunkel found that the Duchenne's gene is huge, relatively speaking, more than 2 million DNA subunits long. Most genes are made up of only 100,000 subunits or less, said McKusick of Johns Hopkins.
The large size of the Duchenne's gene made it impossible to handle with conventional gene-splicing techniques, so Kunkel's group had to break the gene up to handle it in fragments. Each gene fragment was used to make a fragment of the dystrophin protein. The purified protein fragments were then injected into rabbits and sheep to make antibody probes that could recognize the shape of that portion of the dystrophin protein.
These antibodies were used to probe both normal muscle and the muscles of the mdx mice and human patients. In the tests, some of the antibody probes were able to lock onto a protein that was present in normal muscle; but they failed to find the protein in the mdx mice and the human patients.
Now that the protein has been identified, researchers need to produce large amounts of purified dystrophin so its physical characteristics and biological activities can be studied in detail.
It appears that dystrophin is part of a key control region of muscle cells called the triad complex, which like an antenna captures nerve signals and transmits them to muscle fibers, stimulating the muscle to contract.
The triad is made up of membranes from the surface of the muscle cell and the membranes of internal sacs called sarcoplasmic reticulum, or SR, inside the cell. The SR stores calcium, the same mineral used to harden bones and teeth. In muscles, calcium atoms have a different fuction -- to signal protein filaments in the cell to slide past each other and make the muscle contract.
Researchers have yet to identify the precise role of dystrophin in the triad complex, but it appears to play some critical regulatory role, perhaps in controlling the flow of calcium. It's regulatory role is suggested by its large size -- about 400,000 or so molecular weight (hemoglobin, the protein that carries oxygen in the blood is only 64,500) and its relatively low concentration in the cell -- less than 0.002 percent of all muscle protein.
A number of research groups have been trying to understand how calcium is regulated in muscle cells. In a recent report in the Journal of Biological Chemistry, Iowa's Campbell reported discovering microscopic structures in muscle cells called calcium release channels, that serve as protein gates, which direct the flow of calcium out of the SR membranes and into spaces around the muscle filaments, causing them to contract. Initially, researchers thought the calcium release protein and dystrophin were identical because they were similar in molecular size and were both associated with the regulation of calcium in muscle cells. But that proved not to be the case, Campbell said.
In muscular dystrophy patients, the lack of dystrophin alone probably does not make the muscle cell die. Instead, the absence of this protein appears to set off a cascade of effects that ultimately cause the muscle to quite literally fall apart.
There appear to be defects in every muscle function in a Duchenne patient, said Dr. Peter Ray, a geneticist at the Hospital for Sick Children in Toronto and part of a research group also racing to understand the chain of events that results in Duchenne's muscular dystrophy.
One hypothesis, according to Kunkel, is that dystrophin -- in addition to playing a role in calcium regulation -- anchors the protein actin, which with another protein, myosin, make up the filaments in a muscle cell. Without this anchoring effect, muscle fibers cannot regenerate in a normal way.Duchenne's muscular dystrophy usually is not diagnosed until the boy reaches 3 or so because the muscles initially seem to function normally. But as muscle cells become damaged from exercise and growth, they become disorganized and fail to function.
"The Duchenne's muscle can regenerate," Kunkel said. "But the anchor is missing. So it goes into the breakdown process more rapidly."
While Kunkel acknowledges that scientists have a long way to go before they will be able to stop this disease, he believes that the dystrophin discovery is a mark of significant progress.
"The first step was to isolate the gene and use that information to go after the protein," Kunkel said. "Now we have to understand what that protein does and how it can be replaced. And then, finally, address the issue of treatment and whether it is possible or not. You get all the information, and then you design the treatment. It has to be done in a step-wise fashion."