For many people, the announcement that a gene has been found for a disease means that better diagnostic tests are on the way or that gene therapy -- the insertion of a replacement gene -- is close at hand. Although the former is a certainty, the latter is not. Gene therapy is particularly difficult in the neuromuscular diseases, in part because nerve and muscle are such relatively inaccessible tissues (compared to, for example, blood or skin cells).

What gene findings have provided so far for the medical research community is a kind of Rosetta stone for each disease. (The Rosetta stone, found in Egypt in 1799, translated ancient Egyptian hieroglyphics into Greek and allowed scholars to read these symbols for the first time.)

Included below are some gene findings that are now allowing scientists and physicians to understand what's happening in the cells of muscles and nerves affected by genetic disorders. With this understanding, they hope to develop treatments in the not-too-distant future.

HIGH-SPEED TUNNELS DON'T FORM IN X-LINKED CMT

Charcot-Marie-Tooth disease (CMT) is a common disorder of peripheral nerves. In the early 1990s, MDA-supported researchers found three genes that, when mutated, result in the disorder. The genes are all for proteins found in myelin, an insulating sheath that wraps around nerve fibers (axons) and allows nerve signals to travel rapidly from one point to another without leaking out of the fiber.

Now, biophysicist Thaddeus Bargiello at Albert Einstein College of Medicine has described the precise problem that occurs when there are mutations in the gene for connexin 32, one of the myelin proteins. The gene is on the X chromosome. (In X-linked CMT, as in other X-linked disorders, males are more severely affected than females.)

In the October 1997 issue of Neuron, Bargiello's group confirmed what MDA grantees Kenneth Fischbeck and Phillip Chance guessed in 1993: Connexin 32 forms a vital kind of high-speed tunnel known as a "gap junction" in the fibers of the peripheral nervous system.

Without connexin 32, the tunnels don't form properly, the researchers found, leading to what Bargiello calls "big changes in their permeability." Specifically, he notes, one biochemical "major player" known as cyclic AMP won't go through these malformed tunnels.

"If you lose the cyclic AMP signals, we're proposing that you will ultimately lose the myelin," Bargiello says. The signals, he explains, let the myelin-making cells, known as Schwann cells, know that they're in contact with a nerve axon. If they don't get the signals, they give up making myelin and return to a more primitive stage in cell development. They can emerge from this stage, mature again and make myelin again, Bargiello says, but probably only a limited number of times. "Maybe intrinsically Schwann cells can only remyelinate so many times and then they run out of their developmental potential."

Eventually, myelin along the axon is lost, which slows the transmission of the signals from nerve to muscle that allow movement. Also, says Bargiello, without myelin, the axon itself can be damaged. Axons seem to degenerate in this form of CMT (although probably not in all forms). "When you look at nerve biopsies from these patients, you see a lot of atrophy [shrinkage] in the axons and, ultimately, loss of larger diameter axons," Bargiello says. "The eventual loss of axons is probably the factor that leads to the symptoms of muscular weakness associated with the disease."

The challenge now, he notes, is "to understand exactly how the defects in myelin caused by loss of connexin 32's functions result in the loss of these axons."

In their next steps, the researchers will work with recently developed connexin 32 knockout mice, which have no connexin 32. "They appear to provide a pretty good model. They develop a peripheral neuropathy that looks very similar to the human disorder," Bargiello says, "so that these mice will allow us and others working on X-linked CMT to test specific hypotheses."

SMN REVISITED

In a recent issue of Quest (vol. 4, no. 5), we described how new research has fingered the SMN ("survival of motor neurons") protein as the key protein involved in spinal muscular atrophy (SMA). When the SMN gene, which is on chromosome 5, is mutated so that little or no SMN is produced, SMA is the result. It looks like the severity of the disease is roughly correlated with how much SMN is made in the motor neurons.

To make matters a little more complex, the SMN gene comes in two forms on the same chromosome. SMN-C comes from one gene, and SMN-T comes from a gene nearby. SMN-C can partially compensate for the loss of SMN-T. Researchers believe the milder forms of SMA result from the actions of SMN-C, which is present in varying amounts in different people.

Research in SMA and SMN has galloped lately. In our last review of the subject, we were only able to say that SMN is found in structures in the cell nucleus known as "gems." Now, much more is known about SMN, and still more is yet to come, researchers say.

Gideon Dreyfuss is a molecular biologist at the Howard Hughes Medical Institute at the University of Pennsylvania School of Medicine. His career has focused on RNA processing and the transport of proteins and RNA between the cell nucleus (the compartment that houses the DNA on chromosomes) and cytoplasm (main compartment) of cells. Much of the new research has come from his laboratory.

RNA (ribonucleic acid) is the substance formed from the DNA (deoxyribonucleic acid) code in a process known as "transcription." It's the RNA that is eventually transformed into protein in another cellular process known as "translation." When proteins are made in cells, the original code for them is in the DNA, or genes. That code is then transcribed to RNA, which is then processed into smaller RNA (called "messenger RNA"), which eventually leaves the nucleus and enters the cytoplasm. There, proteins are made from the template it forms (see illustration, below).

The original RNA that's transcribed from the DNA code is a kind of "draft," known as pre-RNA. The "draft" molecules of pre-RNA are the same size as the DNA (gene). They carry a lot of excess RNA that doesn't end up in the final code. Pre-RNA has to be processed, says Dreyfuss, who gave a seminar on RNA and SMN at an MDA-sponsored SMA workshop in October. Dreyfuss' group published two papers on the subject in the Sept. 19, 1997, issue of the journal Cell.

SMN, Dreyfuss found, is necessary for the formation of the key "processing workbenches," the spliceosomes. These structures, which are made of specialized RNA molecules and protein molecules, are where pre-RNA is processed to mature RNA.

Assembly of the spliceosomes takes place in the cytoplasm, although they do their RNA processing work in the nucleus. SMN, says Dreyfuss, is a "key spliceosome assembly factor." Without it, spliceosomes don't seem to form. If there's a relative lack of SMN, they probably don't form as effectively. (Specifically, SMN makes it possible for some of the specialized proteins to combine.)

Without spliceosomes, pre-RNA doesn't become mature RNA, and it never makes it out of the nucleus to become the final code for a protein.

So, where do gems come in? "There is also a nuclear pool of SMN," Dreyfuss says. "That pool is concentrated in novel nuclear bodies that graduate student Qing Liu and I described a year and a half ago or so, which we called gems. Therefore, what I described to you so far about the function of SMN in spliceosome assembly is really only the beginning of the story. SMN also has additional functions in the nucleus. We are currently investigating what these functions are."

How Dreyfuss' research group, working in basic cell biology, got to studying SMA is itself a story. A few years ago, the Dreyfuss group had isolated a protein involved in RNA processing. They didn't know the protein they'd found had anything to do with SMA until its genetic sequence was deposited in a data base on the Internet by French researcher Judith Melki as the SMA disease gene.

Just a few years ago, that never would have happened, Dreyfuss says. "This was an Internet connection, definitely." He has since been talking with SMA researchers and clinicians, including MDA grantee Dr. Kenneth Fischbeck at his own institution, the University of Pennsylvania.

"This is a very fundamental finding in cell biology and in molecular biology. But of course for us it is particularly exciting and potentially very rewarding that it linked a very important human genetic disease to a specific biochemical pathway, to a specific biochemical process," Dreyfuss says. "And certainly our hope is that we will understand this process at the molecular level, even at the atomic level, much, much better. If we do, then there's the possibility that we can think of ways to rectify this deficiency [of SMN], to bypass this step, or to increase the efficiency of spliceosome assembly by some other means. So quite a bit of our work now is directed toward that."

MORE ON WAR ON ATAXIAS

In two recent issues of Quest, we described newly discovered mechanisms that underlie the hereditary ataxias, disorders that primarily involve progressive loss of coordination (Vol. 4, no. 5 and no. 6).

MDA grantee Dr. Massimo Pandolfo at the University of Montreal and Montreal's McGill University is among researchers who discovered that Friedreich's ataxia is caused by a lack of the protein frataxin, and that this protein helps keep iron at normal levels in cells. More specifically, it seems to regulate concentrations of iron in the cells' energy-making units, the mitochondria.

Now, researchers at Baylor College of Medicine and the Veterans Affairs Medical Center in Houston have added a new piece to the Friedreich's puzzle. MDA grantees Pragna Patel and Sanjay Bidichandani, with colleague Tetsuo Ashizawa, found that the genetic mutation that underlies Friedreich's ataxia is an unusual kind of mutation, one that seems to change the structure of the DNA, the genetic code.

The mutation in this disorder is an extra-long string of chemicals known as a GAA repeat, which stands for guanine, adenine, adenine. These so-called "triplet repeats" are relatively common in genes that underlie neurologic diseases. In the last few years, about a dozen genetic disorders (most of them neurologic) have been traced to defects concerning such DNA sequences. The problems come when the repeated sections are longer than normal, as if a machine had made too many copies.

In many of the "triplet repeat" disorders that geneticists have so far studied, the repeated section is a CAG repeat, for cytosine, adenine, guanine. This triplet, when it's in the DNA, is a recipe (code) for the amino acid glutamine. In many disorders, including most of the autosomal dominant spinocerebellar ataxias (a group of ataxias that differ from Friedreich's), extra CAG triplet repeats lead to extra glutamine molecules in a cellular protein. This extra-long protein means trouble for the cells affected, but the exact nature of the trouble depends on the protein affected and its normal role and location. (For example, in SCA type 1, abnormal clumps of protein are found in the cell's nucleus, while in SCA type 6, a cellular calcium channel is affected.)

In Friedreich's ataxia, the problem is also a triplet repeat, but there's a catch that has puzzled researchers until now: The extra-long GAA repeats are in a part of the gene that never gets reflected in the final protein. These types of "silent" sections of genes, known as "introns," are common, but their functions are as yet only barely understood. Introns are copied ("transcribed" is the technical word) into the "first draft" RNA code (see illustration above), but they don't make it into the final, completely processed RNA. Why, then, do they affect the final protein -- in this case, frataxin?

The GAA repeat in Friedreich's ataxia has a profound effect on levels of the frataxin protein, because it seems to block the transfer of genetic information from DNA to RNA.

DNA, which is usually in the shape of a double helix (two intertwining spirals), can under some circumstances take on other shapes, such as a triple instead of a double helix. DNA can also loop back on itself or form crosses. With the extra GAA repeats in the DNA, transcription of the frataxin gene into an RNA code is slowed or stopped, and protein production is likewise affected.

The researchers, who published their results in the January 1998 issue of the American Journal of Human Genetics, showed that Friedreich's patients with shorter repeated GAA segments have higher levels of frataxin RNA, and that longer GAA triplet repeats are more difficult to copy into the RNA code. These findings correlate directly with the clear-cut observation of milder disease associated with shorter repeats, according to Bidichandani.

The research team isn't sure exactly what shape DNA takes in Friedreich's ataxia, although laboratory assays tell them that the shape isn't normal. The abnormal DNA may resist unwinding, a necessary part of transcription.

As for the implications of this discovery, the researchers are excited. "We could target the gene defect," Bidichandani says. "It gives us another avenue. So far, everybody's been thinking about strategies to reverse the abnormal iron accumulation. But we could also screen for compounds that would directly affect the gene defect by, for example, destabilizing the DNA structure that forms with the extra repeats. At present, this is a bit premature. We don't really have the technology yet, but there are probably compounds that could act this way."

OPMD GENE FOUND; DEFECT MAY AFFECT CELL NUCLEUS

MDA-supported researcher Dr. Guy Rouleau, a neurologist and neurogeneticist at Montreal General Hospital Research Institute in Canada, was part of an international team that recently identified the gene for oculopharyngeal muscular dystrophy (OPMD). This muscular dystrophy, which usually begins after age 40, causes progressive weakening of the muscles that hold the eyes open and those involved in swallowing. Significant weakness of other muscles, such as limb muscles, occurs in about half of those affected.

The gene, on chromosome 14, codes for a protein that, like SMN, affects the way cells process RNA, a key step in a cell's protein manufacturing process (see "SMN Revisited"). However, the researchers don't think the problem in OPMD is related to a loss of this function. "The protein can still carry out this normal function," Rouleau says, "but it takes on a new, deleterious function in this disorder."

The disease-causing mutation is an expanded "triplet repeat" in the genetic code for the protein known as PABP2 (see "More on War on Ataxias"). In OPMD, the repeat is GCG, standing for guanine, cytosine, guanine in the DNA. GCG is the code for the amino acid known as alanine, a component of many proteins.

In OPMD, extra alanine molecules, caused by extra GCG codes in the gene, are incorporated in the PABP2 protein, where they may cause it to form unusual strands in the nuclei of the affected cells. These strands have been observed in the muscle cells of people with OPMD, but their contribution to the disease process isn't yet clear.

OPMD is found all over the world and is particularly common in French Canadians. Rouleau estimates that one in 1,000 people of French Canadian descent have the potential to get the disorder because they carry the genetic flaw. About 20,000 people in North America have OPMD or may develop it with age.

Rouleau's team considers the findings, published in the February issue of Nature Genetics, unusual and somewhat unanticipated. This is the first triplet repeat disorder known to be caused by the GCG, or alanine, code. Most triplet repeat disorders are caused by the code for glutamine, which is CAG.

Also, the number of extra alanine molecules required to cause the disease is surprisingly few. As little as two extra GCG repeats and therefore two extra alanines can cause the disease, and even one extra repeat can cause it if it's present on both chromosome 14 genes. Disease severity appears to be related to the "dosage" of extra alanines, Rouleau says.

The researchers are already developing experiments toward a gene therapy approach in OPMD, which is now treated with surgery on the eyelid and throat muscles. Current treatments often can't compensate for increasing weakness over time.

GENETIC TESTING FOR SMA

Testing to detect carriers of the SMA genetic mutation on chromosome 5 (the SMN gene) is available through Ohio State University in Columbus. If you desire this type of testing, have your doctor call MDA at (800) 572-1717, and we'll help with connections to the Ohio State research lab.

For genetic testing of people suspected of having SMA, MDA suggests having your doctor call Athena Diagnostics of Worcester, Mass., at (800) 394-4493, extension 9.

REMINDER -- CHECK OUT MDA'S WEB SITE FOR CLINICAL TRIAL UPDATES

If you're looking for the latest information about a clinical trial related to your disorder, the best way to get it is to check out MDA's Web site at www.mda.org.

When you get to the home page, click on Research and then on Active Clinical Trials to see a current list. Additional information about clinical trials can be found by clicking on Links to Major Medical/Research Sites and, when news is breaking, by clicking on What's New on the home page.

There's a lot of other information about research on the Web site as well (see "Window to the World," in this issue), so don't miss out. Don't have the Internet at home? Many public libraries now have Internet access, as do many "cyber cafes" There's probably an Internet connection not too far from you.