Research Updates

• Unique FSHD Mechanism May Open Treatment Doors
• Blocking Myostatin Could Be Effective in DMD
• Mouse Studies Encourage DMD Gene Therapy Search
• Viruses Seen as Factor in DMD, BMD Heart Damage
• Common Link Found for MDs Involving Retardation
• Potent Stem Cells Found in Bone Marrow
• Muscle Stem Cells Help Mice With DMD
• Parents Favor Screening Infants for DMD, Study Says
• Prednisone Might Work Against Congenital MD
• Investigators Studying Neuromuscular Disease With Bone Disease
• Albuterol in DMD Under Study at UCLA
• Hearing Found Normal in Adults With FSHD
• Chemicals Boost Utrophin, Slow Wasting in Mice With Duchenne MD

Unique FSHD Mechanism May Open Treatment Doors

In an MDA-funded study, scientists have determined that facioscapulohumeral muscular dystrophy (FSHD) is caused by a unique mechanism, never before seen in any human disease.

The new study finds that, unlike most forms of muscular dystrophy, FSHD isn't caused by a mutation (error) in a single gene, but by a mutation that triggers the uncontrolled activity of several neighboring genes.

The study, published in the Aug. 9 issue of Cell, "offers new hints for FSHD treatment. It might be possible to design therapies that control the [overactive] genes," lead author Rossella Tupler said.

Under normal circumstances, proteins called transcriptional repressors keep certain chromosome 4 genes inactive, or "turned off." In FSHD, a shortened D4Z4 region on chromosome 4 doesn't allow the repressors to "land" on the chromosome, so the usually inactive genes mistakenly become active, or "turned on."

By 1992, scientists had traced the cause of FSHD to a deletion (a missing piece of DNA) near the end of chromosome 4, in a region that contains repeated blocks of DNA, called D4Z4. Normally, this part of chromosome 4 contains tens to hundreds of D4Z4 repeats, but in people with FSHD, it contains fewer than 10.

Scientists searched for defective genes in the D4Z4 region that might cause FSHD, but found none. Tupler, an Italian scientist who holds appointmentsat the Università degli Studi di Pavia in Italy and the University of Massachusetts Medical School in Worcester, discovered that the D4Z4 region actually represses the activity of genes nearby, and when it's missing — as in FSHD — those genes run amok.

In "a novel research strategy," Tupler's team examined muscle biopsies from people with and without FSHD. They found that in FSHD, at least three genes close to D4Z4 are inappropriately "turned on."

Next, they identified a piece of D4Z4 that acts as a docking site for transcriptional repressors — proteins that attach to DNA and turn off genes. Finally, they showed that by experimentally blocking the repressors in non-FSHD cells, they could reproduce the abnormal gene activity seen in FSHD.

One of the genes turned on in FSHD is ANT1, which is mutated in a form of progressive external ophthalmoplegia (PEO), a disease that weakens muscles around the eyes. Tupler suspects that overactivity of this gene could play a major role in FSHD, which causes weakness mostly in the face, shoulders and upper arms. Overall, "our discovery is a breakthrough toward a complete understanding of FSHD, and opens broad avenues for future research," Tupler said.

Blocking Myostatin Could Be Effective in DMD

In July, at the 10th International Congress on Neuromuscular Diseases in Vancouver (sponsored in part by MDA) two research teams showed that blocking the action of myostatin — a secreted protein that inhibits muscle growth — might be an effective way to treat Duchenne muscular dystrophy (DMD) and other muscle-wasting diseases.

One group, led by Kathryn Wagner at Johns Hopkins University in Baltimore, bred mice lacking myostatin to mice with DMD. The offspring had significantly larger, stronger muscles than DMD mice with intact myostatin, and had less evidence of fibrosis — the replacement of muscle by fat and connective tissue.

In a second study, Tejvir Khurana and his group at the University of Pennsylvania in Philadelphia found they could protect mice against DMD by giving them injections of a protein that blocks myostatin function. The protein is an antibody specially manufactured to target myostatin.

When young mice with DMD were given twice-weekly injections of the antibody, their respiratory muscles showed reduced signs of degeneration compared to those of untreated mice. The treated mice were also larger and stronger, and had lower blood levels of creatine kinase (CK), a protein that leaks out of damaged muscle into the bloodstream.

Wyeth, a biotechnology company based in Collegeville, Pa., developed the anti-myostatin antibodies used in the study by injecting myostatin into myostatin-negative mice and filtering their blood. The company tentatively plans to test the antibodies against age-related muscle wasting and several muscle-wasting diseases, including other forms of muscular dystrophy and diabetes, a Wyeth spokesperson said. But it's likely that human-derived antibodies will be required for clinical use.

Mouse Studies Encourage DMD Gene Therapy Search

Xiao Xiao

MDA grantee Xiao Xiao in the Depart-ment of Genetics and Biochemistry at the University of Pittsburgh School of Medicine was on a team that recently demonstrated that gene therapy for Duchenne muscular dystrophy (DMD) using highly miniaturized dystrophin genes tucked inside a small virus will likely be feasible.

In a paper published in the Aug. 10 issue of Human Gene Therapy, the team found that delivering miniaturized genes for the dystrophin protein, which is missing in DMD, to the leg muscles of mice with the disease, significantly improved the ability of the muscles to generate force and resist contraction-induced damage of the type seen in DMD.

The investigators delivered to the muscles miniaturized dystrophin genes tucked inside adeno-associated viral vectors (transporters). These vectors have demonstrated safety and effectiveness in delivering genes to muscle in previous studies, but their small size makes it necessary to shrink the large dystrophin gene to fit it inside them.

The investigators found that the miniaturized dystrophin gene was still effective in improving muscle function, despite the reduced size of the protein that's made from its instructions.

Jeffrey Chamberlain

MDA grantee Jeffrey Chamberlain, in the Department of Neurology atthe University of Washington Schoolof Medicine in Seattle, published somewhat similar experimental results in the March issue of Nature Medicine (see "Research Updates," April-May 2002). Chamberlain's team found that, when mice with DMD received three types of highly miniaturized dystrophin genes (microdystrophins) tucked inside adeno-associated viral vectors, their leg muscles took on a more normal microscopic appearance. Two of the microdystrophins reversed the signs of muscular dystrophy in the leg muscles, while the third slowed the disease process.

Chamberlain's group didn't address muscle function in the mice treated with the transferred genes but did address it in mice bred with various miniaturized dystrophin genes (transgenic mice). In many of these mice, muscle force generation and injury resistance were better than in untreated mice with DMD, and all were able to run significantly farther on a treadmill than the untreated mice.

Chamberlain's team tested several types of miniaturized genes and found some worked better than others.

Viruses Seen as Factor in DMD, BMD Heart Damage

Viruses may be a major contributor to the type of heart problem seen in Duchenne and Becker muscular dystrophies (DMD and BMD), say investigators at the University of California at San Diego.

In a paper published in the August issue of Nature Medicine, Dingding Xiong and colleagues show that a lack of dystrophin, the protein that's absent or disrupted in DMD and BMD, makes the heart more susceptible to infection with at least some viruses, particularly the coxsackie virus. They conclude that, while there are probably a number of mechanisms by which dystrophin deficiency leads to destruction of the heart muscle — a problem known as cardiomyopathy — their data support the idea that dystrophin deficiency increases susceptibility to virally induced heart muscle damage. (For more on cardiomyopathy in DMD and BMD, see "The Heart Is a Muscle Too," Quest, vol. 6, no. 2, 1999.)

Common Link Found for MDs Involving Retardation

Certain forms of muscular dystrophy are associated with mental retardation, but the reason for this pairing has been elusive.

In two new studies, scientists funded by MDA have found evidence that a protein called alpha-dystroglycan is at the root of both the muscle and brain abnormalities in these diseases. The finding has implications for treating some forms of MD and mental retardation, whether they occur together or alone.

Kevin Campbell

"As we build a case for alpha-dystroglycan being a major player in these diseases, it allows us to think about global therapeutic strategies that could work for all of them," said lead scientist Kevin Campbell, an investigator at the Howard Hughes Medical Institute and a professor in the Departments of Physiology and Biophysics, and Neurology, at the University of Iowa in Iowa City.

Some forms of congenital (infantile-onset) MD — Fukuyama CMD, muscle-eye-brain disease (MEB) and Walker-Warburg syndrome (WWS) — are strongly associated with severe mental retardation. Duchenne muscular dystrophy is often associated with learning disabilities.

Based on recent evidence that FCMD and MEB are caused by defects in glycosylation (the process of adding sugars ontoproteins), Campbell and his team examined alpha-dystroglycanin muscle biopsies from children with these diseases. They found that most of the alpha-dystroglycan didn't have its normal sugar components and therefore wasn't able to interact with other proteins found in brain and muscle. An animal model of CMD, the myodystrophy mouse, also lacked the normal form of alpha-dystroglycan, and had brain abnormalities reminiscent of FCMD, MEB and WWS.

In a second study, Campbell and his team deleted the dystroglycan gene in the brains of otherwise normal mice. Those mice also developed brain abnormalities similar to those seen in FCMD, MEBand WWS. The two studies appeared back-to-back in the July 25 issue of Nature.

Campbell said the findings also could have implications for DMD, but that more research needs to be done. "We're going to see if we can make a stronger case for an involvement of alpha-dystroglycan in the Duchenne learning abnormalities," he said.

He also cited evidence that alpha-dystroglycan might play a role in some forms of mental retardation not tied to muscular dystrophy. In a study last year, his group showed that alpha-dystroglycan serves as a binding site for LCMV, a virus that can trigger brain abnormalities during fetal development.

Campbell and his group are developing gene therapy tools that could be used to treat CMDs. "It's still early, but our results present a new set of therapeutic strategies that can be pursued in the laboratory," he said.

Potent Stem Cells Found in Bone Marrow

A new study, published online by Nature on June 23, strengthens the possibility of using bone marrow-derived stem cells to treat neuromuscular diseases.

Catherine Verfaillie and colleagues at the University of Minnesota in Minneapolis isolated stem cells from the bone marrow of adult rodents, and found some with remarkable similarity to embryonic stem cells. The cells — called multipotent adult progenitor cells, or MAPCs — can divide for many generations in a culture dish, and with some coaxing, they can produce many cell types, including nerve and muscle.

Moreover, when just one MAPC is implanted into an early rodent embryo, it can give rise to progeny in tissues and organs throughout the adult animal, in-cluding brain and muscle.

However, when Verfaillie and her group gave adult rodents intravenous injections of MAPCs, the cells only contributed to blood and other tissues that are rapidly turned over — not to muscle or brain. Further research will determine whether MAPCs can home in on and repair muscle or brain tissue that's been damaged by disease.

Muscle Stem Cells Help Mice With DMD

MDA grantee Johnny Huard, a cell biologist at the University of Pittsburgh, was on a team that recently isolated a new type of stem cells from the muscles of healthy mice and transplanted them into mdx mice, which have Duchenne muscular dystrophy (DMD). The findings are published in the May 27 issue of the Journal of Cell Biology.

Johnny Huard

The newly isolated cells have the ability to turn into muscle, nerve or endothelial cells (which line the blood and lymph vessels and some body cavities). When the cells were transplanted into mdx mice, numerous muscle fibers containing the dystrophin protein, missing in those with DMD and in mdx mice, appeared.

The results suggest that some of the previous obstacles encountered with muscle cell transplantation — low survival rate of the transplanted cells, poor spreading of the cells and rejection of the cells by the immune system — might be overcome by using this new type of cell, assuming that such cells have parallels in the human body.

Parents Favor Screening Infants for DMD, Study Says

Carol Hoffman, a graduate student in genetic counseling at Brandeis University in Waltham, Mass., recently conducted a survey of parents of children with Duchenne muscular dystrophy (DMD) and found that a large majority favor screening infants at birth for potential early diagnosis of DMD.

Ninety-three parents were included in the final analysis. The survey results showed:

• The average age of diagnosis for DMD in the United States was 4 years, 5 months. A delay in diagnosis most often occurred when children came to medical professionals as infants or toddlers or when primary care physicians initially took no action.
• 86 (92.4 percent) of respondents were in favor of some form of infant screening; 59 (63.4 percent) thought screening should be voluntary, while 27 (29 percent) thought it should be mandatory. One person thought infant screening for DMD shouldn't be performed at all, and six people had no opinion.
• If screening were to be implemented in the United States, the program should probably be voluntary, emphasizing parental choice and informed consent.
• Any screening program should involve an integrated team of health professionals to provide continuing education and support for families.
• Early diagnosis can have negative consequences (e.g., loss of "not knowing," prolonged distress, etc.). Parents who choose to undergo infant screening should be made aware of these potential effects before embarking on the testing.
• Infant screening can't alleviate the distress and frustration associated with lack of sensitivity, psychosocial support, resources or communication parents may encounter, or the shock and trauma of receiving the diagnosis.

Prednisone Might Work Against Congenital MD

Prednisone, a steroid drug with anti-inflammatory effects, is often prescribed to slow the course of Duchenne MD. A new MDA-funded study suggests that it and other anti-inflammatories also might work against the most common form of congenital muscular dystrophy, merosin-negative CMD.

Merosin-negative CMD, which results from a deficiency of the protein merosin (also called laminin-alpha-2), causes slowly progressive muscle weakness and wasting, often noticeable at birth. There's evidence that inflammation plays a role in the disease.

Prednisone and other corticosteroids suppress inflammation by acting directly on blood-borne immune cells, and by blocking complement — a set of proteins in the blood that stimulates the activity of immune cells. More specific complement inhibitor drugs are under development.

In the new study, Anne Connolly and her colleagues at Washington University School of Medicine in St. Louis tested the effects of prednisone and complement inhibition on mice with merosin-negative CMD. She found that mice lacking the gene for C3, a complement protein, lived longer than those with normal C3. When treated with prednisolone (a liquid form of prednisone), these mice showed even greater increases in survival and strength.

"I consider this encouraging news, but it would take a clinical trial to see if [these drugs] are effective for children with CMD," Connolly said of the study, published in June in the Journal of Neuroimmunology.

Investigators Studying Neuromuscular Disease With Bone Disease

Investigators Virginia Kimonis and Giles Watts at Harvard University Med-ical School in Boston are seeking families with a history of neuromuscular disease combined with inclusion bodies and Paget disease of bone.

Inclusion bodies are cellular abnormalities that can be detected in a muscle biopsy; Paget disease is associated with pain in the bones and characteristic X-ray findings.

The team has identified five large families with this combination of symptoms, which doesn't yet have a specific disease name. About one-third of the family members also exhibit frontotemporal dementia, which involves language deficits, personality changes and other cognitive problems.

The group has found a region on chromosome 9 that may hold the gene underlying this complex disease. More families are needed to pin down the specific gene involved.

The disease may have been diagnosed as limb-girdle or facioscapulohumeral muscular dystrophy, inclusion-body myopathy or ALS-like disease.

If you've received a diagnosis of, or have a family history of, a muscle disease with inclusion bodies and have Paget disease of bone, the researchers would like to hear from you and will travel to your home. The only invasive procedure is a blood test.

For information, contact Kimonis or Watts at (617) 355-4697, (617) 355-7748 or virginia.kimonis@tch.harvard.edu.

Albuterol in DMD Under Study at UCLA

MDA grantee Melissa Spencer at the University of California at Los Angeles and colleagues are studying the drug albuterol in Duchenne muscular dystrophy (DMD).

Albuterol, which is usually used to dilate lung passages in respiratory diseases, may increase muscle mass and strength. When used for muscle disease, the drug is given orally in a slow-release tablet.

Researchers believe albuterol may interfere with the actions of calpain, a protein that could play a role in muscle destruction.

A preliminary trial of albuterol in 10 boys with DMD found a significant increase in average strength. The re-searchers are now conducting a double-blind study with 25 to 30 boys with DMD.

"In our initial study we had very few side effects in the patients," Spencer said. "Our hope is that albuterol treatment will become a component of an armamentarium of drugs that together will reduce the progression of DMD until a permanent cure is identified."

Eight visits to UCLA over the course of nine months will be required. The boys will undergo DEXA scans, low-dose X-rays that measure lean body mass (bone and muscle density).

For information, contact Linda De Sepulveda at (310)825-9816 or lbdesepu@ucla.edu; or Melissa Spencer at (310) 794-5225 or mspencer@mednet.ucla.edu. For more information, go to www.physci.ucla.edu/DMD/albuterol.html.

Hearing Found Normal in Adults With FSHD

A new study by Mark Rogers and colleagues at University Hospital of Wales in Cardiff overturns earlier reports that have suggested hearing loss may be part of facioscapulohumeral muscular dystrophy (FSHD).

The study, published in the May issue of Neuromuscular Disorders, examined 21 people with adult-onset FSHD and compared the results with data obtained from a British National Study of Hearing, which covered the general population.

Much to the investigators' surprise, hearing in those with adult-onset FSHD was actually better than that of the general population, although the differences were statistically significant only on some measures.

The authors suggest that hearing impairment may be an important component of infantile- or childhood-onset FSHD but that it appears not to be part of adult-onset FSHD. They caution that their numbers are small and that further studies on larger populations need to be performed to verify their findings.

Chemicals Boost Utrophin, Slow Wasting in Mice With Duchenne MD

In experiments on mice, scientists funded by MDA have identified two chemicals that can partially compensate for the loss of dystrophin, the protein missing in Duchenne muscular dystrophy (DMD). Both chemicals appear to work by boosting the production of dystrophin's sister protein utrophin.

Two groups presented the findings in July at the 10th International Congress on Neuromuscular Diseases in Vancouver, partly sponsored by MDA.

One group, led by Sabine de la Porte of the Centre National de la Recherche Scientifique in Gif-sur-Yvette, France, found that the small compound L-arginine can increase utrophin levels in mice with DMD. Another group, led by Tejvir Khurana at the University of Pennsylvania in Philadelphia, found that a small protein called heregulin has a similar effect.

When injected into the abdominal cavities of mice with DMD, each chemical partly protected against the muscle wasting associated with the disease.

Scientists have been searching for ways to stimulate the production of utrophin since its discovery in 1989. De la Porte decided to investigate L-arginine because it's a source of "fuel" for nitric oxide synthase (NOS), a protein that generates the small signaling compound nitric oxide and is closely linked to utrophin at the muscle membrane.

After several months of L-arginine, given once daily for five days a week, the limb and respiratory muscles of mice showed fewer signs of degeneration, and their respiratory muscles showed improved strength, compared to those of untreated mice. Treated mice also had lower blood levels of creatine kinase, a protein that leaks out of damaged muscle cells.

During years spent studying the gene that encodes utrophin, Khurana and his group learned the gene could be "turned on" by heregulin, a protein that nerves release to stimulate muscle development. The limb muscles of DMD mice that were given twice-weekly heregulin injections over a three-month period showed less degeneration and improved resistance to contraction-induced damage compared to those of untreated mice.

It's not yet clear if L-arginine or heregulin will be useful in children with DMD. De la Porte acknowledged that because nitric oxide controls many processes in the body, including blood vessel dilation and cell metabolism, L-arginine becomes toxic with increasing dosage. The potential toxicity of heregulin at high doses hasn't yet been addressed.

It's also unclear whether either of these proteins could "upregulate" or increase utrophin levels to the extent necessary to protect muscles completely against DMD.