Family Histories Help Solve Medical Mysteries
People with Myotonic Dystrophy Spur Research Advances

The year was 1992, and neurologist John Day had recently moved from the University of California at San Francisco to the University of Minnesota at Minneapolis, where he was to assume the directorship of the MDA clinic. (Day still holds this position, at what is now the Fairview-University Medical Center. He's also an associate professor of neurology at the university.)

Earlier that year, a genetic defect underlying myotonic muscular dystrophy (MMD) had been identified by MDA-supported researchers, and a genetic test for it had just been made available.

The MMD genetic defect, located on chromosome 19, was an unusual one that had rocked the scientific community in the early ‘90s. It consisted of a series of repeated segments of DNA that could actually expand as they were passed from parent to child. As they did so, the disease appeared to worsen in the next generation, sometimes dramatically so.

The segments were made up of three chemicals known as DNA bases — cytosine (C), thymine (T) and guanine (G) — so they were called CTG repeats, triplet repeats or trinucleotide repeats.

In addition to the stir caused by the unstable nature of the DNA change, or mutation, there was another mystery to the chromosome 19 MMD discovery. The genetic flaw, located inside a gene that came to be known as DMPK, was in a part of the gene that shouldn't have mattered.

Previously, mutations known to lead to genetic diseases had almost always been found in the "coding region" of a gene — the part that carries instructions for making a protein. The DMPK mutation underlying MMD wasn't in a coding region, but rather in a part of the gene that carried no instructions and seemed to have no specific function.

Even so, by late 1992, it seemed that the unusual mutation on chromosome 19 was responsible for all of the many symptoms that can occur in MMD, the most common form of muscular dystrophy affecting adults. Among its signs and symptoms are myotonia, which means difficulty relaxing muscles at will; cataracts in the lens of the eye; weakness, especially in certain muscles, such as those of the lower legs, forearms, hands, neck and face; respiratory and cardiac abnormalities; gastrointestinal problems; excessive sleepiness; hormonal irregularities; relative insensitivity to insulin; and even a personality type that some experts have described as apathetic, unconcerned or lacking in emotion. The number of repeated CTG segments in a person was roughly correlated with the number and severity of MMD symptoms.

Charles Thornton, an MDA research grantee and clinic co-director at the University of Rochester Medical Center in upstate New York, remembers the finding as "the most exciting scientific development during my professional life."

Like Thornton, John Day was grateful to have a genetic test that could provide a clear diagnosis of MMD for his patients and, at least to some extent, predict its severity.

A Curious Difference

A few months after arriving in Minnesota, Day saw six members of a large family who were affected by myotonic dystrophy. "I said, ‘That's interesting; let's get a gene test' — and it came back negative."

As Day was puzzling over why the genetic tests didn't show the chromosome 19 defect, a woman came to see him in the clinic. She was expecting a child, knew there was a form of muscular dystrophy in her family, and wanted information.

"I remember sitting there talking with her," Day says. "And I said, ‘Have you ever had any symptoms?' She answered, ‘Driving down here today, I couldn't let go of the steering wheel. Things like that happen to me all the time.'"

Day remembers that his "heart kind of sank." The symptom she was describing sounded like myotonia, which might well mean she had at least a mild form of MMD. He also knew — as the 1992 gene identification studies had clarified — that a woman with even very mild MMD could give birth to a severely affected child, one with the so-called congenital form of MMD, which involves mental retardation, very weak muscles, difficulty sucking and swallowing, and trouble breathing.

Day anxiously sent her blood to the lab and waited. It, too, came back negative for the genetic mutation on chromosome 19. Day says, "Now I was really getting perplexed."

Even the testing center at Baylor College of Medicine in Houston began to wonder about the Minnesota diagnosticians, Day recalls. "I think they were wondering what we were up to up here. Within a matter of months, we had identified three or four separate families who had been diagnosed with myotonic dystrophy and yet came up genetically negative."

Collaboration

Day suspected there was another gene that could, when flawed, lead to myotonic dystrophy — or something that looked very much like it.

He wasn't the only one who thought so. By the mid-1990s, investigators at the University of Rochester and in Germany each had identified a group of people who had what they called proximal myotonic myopathy, or PROMM, a disorder that closely resembled the chromosome 19 form of MMD with one exception: Instead of the muscle weakness landing mostly on the muscles farthest from the center of the body (the distal muscles), these patients had more weakness in muscles closer to the body's center (the proximal muscles). Instead of having difficulty using their hands and wrists, for example, they tended to have more trouble climbing stairs and getting up from chairs.

Then, in 1994, Day attended a lecture by molecular biologist Laura Ranum, who was at the University of Minnesota studying the genetic basis of a neurologic disease called spinocerebellar ataxia type 5, an inherited form of incoordination. (Ranum received MDA funding for this work and later for MMD research. She's now in the university's Department of Genetics, Cell Biology and Development and, like Day, is part of the Institute of Human Genetics.)

Clearly, Ranum was an expert gene hunter and had a keen interest in neuromuscular disorders as well. It wasn't long before Day encountered her coming in from the university parking lot and struck up a conversation about the unusual MMD patients whose genetic tests were negative for the known mutation.

With seed money from the university and later major support from MDA, the two began a fruitful collaboration to identify what they both suspected was at least one additional genetic flaw underlying MMD.

Field Trips

"An investigation depends on two things — a family that has dedicated themselves to figuring out what's going on, and researchers who are sufficiently interested, motivated and funded to do the investigations," Day says. Fortunately, he and Ranum had both.

MAKING MEDICAL HISTORY IN MINNESOTA
Wendy Salo's doctors told her she was suffering from stress — or perhaps chronic fatigue syndrome. But Salo knew there was something else going on. At the time, she was in her late 30s. Admittedly, she had reason to be tired and stressed. She had a full-time job developing software for banks, and she had a husband and two teen-age sons. But, to her way of thinking, those factors couldn't account for her nearly Wendy Salo and her dog, Chumly	falling asleep at the wheel during the 15-minute drive between work and home, or being unable to climb the stairs to her office after her lunch break. "My legs wouldn't do the normal stair motion. They just locked up," says Salo, now 42. Just when she was reaching her limit with frustration and exhaustion, Salo traveled to northern Minnesota for a family funeral. Her father's brother had died, and Salo found herself engaged in conversation with his daughter. They found they shared many of the same symptoms, as had Salo's uncle. Salo's next step involved a trip to Minneapolis, where she saw MDA clinic director John Day — and began to get some answers. When she and other family members tested negative for the known cause of	myotonic dystrophy, they quickly became involved in Day's investigations to find a second genetic cause of the disease. Day took advantage of Salo's parents' 50th wedding anniversary in the summer of 1999 to test several family members and to give some of them, including Salo, a diagnosis at last: the chromosome 3 form of myotonic dystrophy. Salo's fatigue has been alleviated by an over-the-counter supplement she takes with Day's supervision, and her frustration and anxiety have been much relieved by having a definite diagnosis. She's adjusted to using a different type of computer keyboard and has given up skiing. But, this winter, she's planning trips to Guatemala and Mexico, for which she's learning Spanish. "If you can't do something anymore," she says, "[replace] it with something you can do."

A large family in which the mysterious form of myotonic dystrophy was prevalent was ready and willing to help the university team. "This family," Day says, "was one of those families that really wanted to invest themselves in figuring out the disease." (They have, however, requested anonymity.) Ranum, Day and their colleagues were equally enthusiastic.

"There were probably six of us," Day recalls of their first field trip to rural Minnesota in the spring of 1995. The team, consisting of Ranum, Day, nurse practitioner Sandy Whitmore (who was also Ranum's sister-in-law), various technicians and genetic counselors, and an MDA services coordinator piled into a university van and drove some five hours to a town so small it was hard to find sweatshirts to buy to protect them from the late spring chill.

They set up a makeshift "clinic" in a church the family was using for a wedding and reunion. Stations were constructed where they could do basic neurological exams, electromyograms (EMGs) to check for myotonia and eye exams to check for cataracts; take family histories and construct family trees; and, of course, take the important blood samples for DNA testing, which technicians ferried back to Minneapolis in shifts.

"The groom was great in allowing us to do this," Ranum recalls. "It didn't seem to faze the family. They had been dealing with this [disorder] a long time."

To "map" a gene, Day explains, "you start out looking at a whole lot of different [genetic] markers and seeing whether or not there's any particular marker that tends to be present in affected versus unaffected individuals. Once you find a marker that tends to cosegregate [go along with] the disease in a family, you know you're on the right page."

There would be several more trips over the next few years, especially for Day, who tracked down relatives of the family then living as far away as Texas and Alaska.

German Connections

Then, in 1997, Ranum and Day attended an international workshop on PROMM in Naarden, the Netherlands. The meeting was small, and the 20 or so researchers ate, drank, met and talked with each other.

Among the participants was Ken Ricker, a neurologist from the University of Wurzburg in Germany, who had identified many families in his country who appeared to have PROMM. But the families weren't large enough for the genetic linkage studies that lead to finding a gene.

Peter Harper, a physician and medical geneticist at the University of Cardiff in Wales and a longtime expert in MMD, recalls this period. "Because [Ricker] was an electrophysiologist," Harper explains, "he had anybody with a sort of funny disorder with a little myotonia from all over Europe sent to him. So he had this great battery of atypical patients." Out of that battery, says Harper, he had recognized "a definite group."

Ranum and Day wondered whether Ricker was seeing the same disorder they were seeing in Minnesota. If so, combining their experience would be helpful for all concerned.

By the following year, Ranum and Day and colleagues had mapped the Minnesota MMD gene to chromosome 3 and traveled to another meeting, this time in Adelaide, Australia, to present their findings. Again, Ricker was there.

"That's when we really started talking about collaborating on this," Day recalls. "It was really a very strong collaboration, because there was a lot that each group could bring to the other, a lot of complementary resources."

Ricker joined the Minneapolis team on their next field trip, this time to St. Cloud, Minn. Ranum remembers the trip fondly. To Ricker, she says, the large, multigenerational Minnesota families were "just amazing." In Germany, families tended to be small and had been disrupted by war and other conditions that caused them to disperse. In Minnesota, family members had mostly stayed near each other, an ideal setup for a genetic study.

Meanwhile, Ricker's German families showed a genetic flaw at the same location on chromosome 3. But it wasn't yet clear whether there was more than one gene involved, or more than one mutation in the same gene leading to two different disorders. The prediction, Ranum recalls, was that two separate genes would be found to underlie the disorder in the German and Minnesota families.

After holding another improvised clinic in St. Cloud, Ranum, Day, Ricker and graduate student Christina Liquori were looking at the moon when Liquori said, "Look, a diamond." Ranum thought she was referring to a celestial event, but in reality the student had found an actual diamond in the parking lot — which she turned in for a reward. It seemed to be a good omen for the needle-in-a-haystack gene search that was nearing its end.

GETTING BY IN MISSISSIPPI Sheila Smith of Brookhaven, Miss., can't get over the feeling that myotonic dystrophy is an unwelcome, and unexpected, intruder in her family. It started in November 1998, when her husband, Michael, then 33, had a car accident. "We feel that he fell asleep at the wheel," Sheila says. Sheila and Michael Smith with their children, Mick and Whitney. In the months leading up to the accident, Michael had learned he had cataracts and had surgery to remove them, and he'd developed some other disturbing symptoms. He'd shown signs of sleep apnea, a periodic cessation of breathing during sleep, and kept falling asleep during the day. His hands, always weak (something he and Sheila attributed to football injuries), had lost so much strength that he'd had to give up his career as a truck driver. Thinking a desk job would be better, Michael took up telemarketing, but that, too, proved unworkable as his speech became increasingly slurred. While Michael's injuries from the car accident were serious, doctors predicted a good recovery. They told Sheila that her husband had broken some ribs and injured his spleen, and that his lungs appeared "bruised." They were placing him on a ventilator as a temporary measure while his injuries healed. But Michael couldn't be weaned from the ventilator to breathe on his own. Further tests revealed the source of many of Michael's troubles, including his respiratory problem: He had myotonic dystrophy. Further investigation revealed that Michael's entire family was affected by the disorder, including the congenital form in a daughter of his cousin. Those in the immediate family had their lives turned inside out. Sheila gave up her career plans to take care of Michael. "I feel I'm a self-made nurse," she says of caring for her ventilator-dependent husband around the clock in their mobile home. After two years with little or no help, Michael became eligible for Medicare's disability program, which provides some nursing care. Changes in Michael's ventilation system (he now uses a bilevel pressure system delivered through a tracheostomy) and improvements to his cardiac care have made a difference. "He was a totally new person," Sheila says of the improved ventilator setup. "There's no more drowsiness. It's been a wonderful turnaround." Michael, now 36, spends his time reading adventure stories by Louis L'Amour and "aggravating my kids." His family, he says, are his life now, and because of them, he's "not ready to give up." Sheila worries about the health of her two children. "If we had known this was in our family, we would have elected not to have children," she says. Greater awareness of the disease, she says, would help everyone — at least until there's a cure. The Smiths have decided to wait until the children are old enough to make their own decisions about genetic testing for myotonic dystrophy (a policy upheld by many U.S. medical centers). Back row, from left: Mark Smith, Larry Smith and Matthew Smith. Front row, from left: Gretchen Brumfield, Michael Smith and Elana Brumfield. Larry and his three sons, Mark, Matthew and Michael, and their cousin, Gretchen, have myotonic dystrophy. Gretchen's daughter Elana has the congenital form.

Eureka

It was Thanksgiving Day 2000. Liquori was curious about some genetic tests she had been running and decided to come into the lab to check on some results.

Thinking she saw some important differences in the suspect area of chromosome 3 between the affected and unaffected family members, but not wanting to disturb anyone's holiday, she sent an e-mail to Ranum — who didn't see it until Sunday.

"When I saw the e-mail, I went in," Ranum remembers. She agreed with Liquori: There was something different between the affected and unaffected DNA. It might be the mutation itself.

"I paged John [Day], and he called from the grocery store. I was ecstatic. John was ecstatic." Liquori, says Ranum, was calm — calm enough to have "managed not to call us over the weekend."

The Thanksgiving eureka, for all the ecstasy it produced among the researchers, was not yet a "publishable result," Day recalls. "We had to see if it was really present in all the affected individuals — and to see if it was present in unaffected people, too. And, what the heck was it? It was a matter of purifying it and characterizing it."

WHAT REALLY CAUSES MYOTONIC DYSTROPHY? Normally, proteins are made following a DNA "recipe." First, the DNA is converted to its close chemical cousin, RNA, in the cell nucleus. The RNA undergoes further processing and is then transported out of the nucleus and into the cell's main compartment (cytoplasm), where it's then used as an instruction manual to make a protein (A). In both forms of MMD, extra DNA in a gene leads to extra RNA (B). The extra RNA is thought to be too big to leave the cell's nucleus, so its protein can't be made (1). Experiments in the early 1990s tested the theory that the chromosome 19 gene with extra DNA repeats (the cause of type 1 MMD) would keep DMPK from being made into a protein. They showed that the DMPK protein levels are about 50 percent below normal in people with MMD. Mice bred to lack DMPK show some skeletal muscle and cardiac problems, but they don't show the full range of MMD symptoms, so lack of DMPK was ruled out as the sole cause of MMD. Whether it's a contributing factor is still unknown. Later experiments found that genes near DMPK might also be affected by the expanded DNA — a sort of "neighborhood" effect on genes in the region of the mutation (2). Studies in mice lacking SIX5, a protein normally made from a gene near the DMPK gene, have shown that these mice develop cataracts, a sign of MMD, supporting the idea of genetic "neighborhood" effects. Today, researchers believe these effects could account for some of the differences between type 1 and type 2 MMD. After August 2001, when the gene was found for type 2 MMD, attention shifted to the expanded RNA and how it disrupts operations in the nucleus in both forms of the disease (3). CELL COMPARTMENTS NORMAL GENE PROCESSING WITH EXTRA DNA

As the researchers had hoped, the newly identified difference turned out to be present only in people with what would soon be known the world over as type 2 myotonic dystrophy.

Much to everyone's surprise, the German families and upstate New York families, previously thought to have different mutations leading to PROMM, had exactly the same mutation. The differences in the proximal versus distal weakness hadn't amounted to much after all. (Some experts believe the differences in the weakness pattern are real and important and that further research should be done to see what causes them, while Ranum and Day believe the differences may be at least partly in the eyes of the examiner rather than in the patients themselves.)

Although the term PROMM is still used to describe symptoms, the official terminology for myotonic dystrophy is now "type 1" for the chromosome 19 form and "type 2" for the chromosome 3 form. (Doctors abbreviate these DM1 and DM2, for "dystrophia myotonica," another name for myotonic dystrophy. MDA uses the MMD abbreviation to avoid confusion with dermatomyositis, which is labeled DM.)

More Surprises

After the discovery of the chromosome 3 form of MMD (published in the Aug. 3, 2001, issue of Science), there were still more surprises to come.

The mutation on chromosome 3 was found to be a quadruplet repeat — a sequence of four DNA chemicals repeated up to thousands of times, far above the normal number. This time the sequence was CCTG — two cytosines, a thymine and a guanine DNA base.

The CCTG repeats are located in a gene called ZNF9. And, like the CTG triplet repeats in type 1 MMD, the type 2 repeats are in a region of the gene that doesn't provide instructions for a protein — yet they lead to a disease that clearly affects many proteins.

It became clear to researchers that if the two forms of MMD show almost exactly the same group of symptoms, but arise from defects on different chromosomes, the effects of the flaws can't be limited to chromosome-specific, or "local," effects. The disorders must have a common molecular origin, probably related to the effects of the mutations on operations in the cell nucleus.

Laura Ranum and others say the similarities between the two forms of MMD and the mechanisms underlying those similarities will probably take center stage in developing treatment strategies. The similarities are likely related to what happens to RNA — the chemical to which DNA is converted in the cell nucleus (see "What Really Causes Myotonic Dystrophy?").

RNA that's too long, recent studies have shown, can throw a wrench into the workings of a cell's nucleus, disrupting the activities of one or more proteins that have important jobs there. Since both forms of MMD probably have this type of RNA disruption, this factor is now the front-runner to explain these diseases. And this "toxic RNA" is the lead target in the quest to find treatments for MMD.

Ranum, for one, is optimistic about an eventual treatment strategy. "If the RNA mechanism has a limited number of other proteins that are involved, you might be able to figure out some way to control that," she says. An important step is finding a mouse model in which to test ways of targeting specific proteins.

Why the Differences?

However, the differences between the two types of MMD are important. So far, the severe, congenital-onset form associated with type 1 hasn't been seen in type 2. It isn't clear that the chromosome 3 disease worsens as the gene "grows" or is passed to a new generation. And, the apathetic personality type sometimes associated with type 1 MMD apparently isn't part of type 2.

Laura Ranum (front left) and John Day (front right) and their lab staff at the University of Minnesota. Christina Liquori is in the red sweater, middle right.

Ranum believes the differences may have to do with the effects of the two mutations on surrounding genes on their respective chromosomes.

Some researchers believe type 2 MMD only amounts to about 2 percent of the total number of people with myotonic dystrophy, but, since genetic testing on a commercial basis doesn't yet exist for this form of the disease, it's hard to estimate its true prevalence.

Ranum, however, thinks type 2 may have been vastly undercounted. So far, it seems to affect mostly those who can trace their ancestry to Germany, Poland or eastern Russia. Type 1 seems to be far more common in the general population. Why the German families seem to have more proximal weakness than the Minnesota families, even though they share the same genetic flaw, remains to be studied.

Optimism

Doctors at MDA clinics are already working on reducing the myotonia in MMD with various medications. They're treating the cardiac problems with drugs and sometimes pacemakers, and they can alleviate some of the sleepiness, gastrointestinal symptoms and problems related to insulin insensitivity.

But few of the drugs or treatments now being studied are expected to actually reverse or cure either form of the disease.

For that, doctors agree, you need to get at the molecular problem itself — a prospect thought daunting a decade ago, but one that now seems almost within reach.

During the last decade, Peter Harper says, experts in the field "have been rather fixated on the basic science." Now, he says, "they're starting to swing back to management and the clinical side of things."

The mood among his research colleagues, he says, is "pretty optimistic."