An unusual dog, a new approach to muscular dystrophy: Stimulating a protective gene

muscular dystrophy
Vieira with Ringo

Ringo was a golden retriever that defied the odds. Despite having the gene mutation for Duchenne muscular dystrophy (DMD), he remained healthy. And he’s provided a new lead for boosting muscle strength in DMD, one of the most common forms of muscular dystrophy. Unlike other dogs with the dystrophin mutation, who are weak and typically die by 2 years of age, Ringo was able to walk and run normally and lived to the age of 11, within the normal range for golden retrievers.

What made Ringo so resilient? A collaborative study by Boston Children’s Hospital, the Broad Institute of MIT and Harvard and the University of São Paulo in Brazil, published in Cell last week, found that he had a protective gene that boosts muscle regeneration, helping him “escape” the effects of the dystrophin mutation. His son Suflair has it too.

The Boston Children’s lab of Lou Kunkel, PhD, which has a collaborative research agreement with Pfizer, is now using a zebrafish model of DMD to screen for drugs with the same protective effect.

muscular dystrophy Jagged1 counteracts dystrophin mutation in dogs
Suflair (at right) with two severely affected dogs

A dogged search

Natássia Vieira, PhD, a fellow in Kunkel’s lab from the University of São Paulo and first author on the Cell paper, had been studying a colony of golden retrievers in Brazil that had the classic DMD mutation, which causes loss or dysfunction of the dystrophin protein, which helps hold muscle together. (Healthy dogs from the colony are trained as service dogs for children with muscular dystrophy.)

Ringo, aside from being surprisingly active, also proved adept at siring puppies — 49 with four different females. “When people would leave the door open he would run and mate,” Mayana Zatz, DSc, of the University of São Paulo and co-senior author on the paper with Kunkel, told Nature News.

Natassia Vieira and Louis Kunkel Jagged 1 muscular dystrophy

When Vieira joined Kunkel’s lab as a postdoc, they set out to find out why Ringo and Suflair had fully functioning muscle, even without dystrophin. “We decided to do genome-wide association studies (GWAS) to see where in the genome there might be a gene that modifies disease severity,” says Kunkel.

Kunkel and Vieira decided to partner with Kerstin Lindblad-Toh, PhD, at the Broad Institute, who had led the Institute’s DogDNA project and was intimately familiar with their genetic complexity.

“I wanted to knock on her door because it’s really hard to work with dogs,” says Vieira. Dogs have highly diverse genomes, she notes, and each breed has a different genomic signature.

Combining family linkage analysis and GWAS, the team compared the genomes of Ringo and Suflair with those of 31 severely affected golden retrievers. They found that a region on chromosome 24 tracked with disease severity.

To narrow the search, Vieira then used gene expression arrays, which measure what genes in a DNA sample are expressed (turned on). When she compared Ringo and Suflair with the affected dogs, she found 65 genes that were differently expressed. But only one gene was on the region of chromosome 24 flagged by the GWAS study: Jagged1, a gene known to be involved in muscle regeneration.

But what change in Jagged1 accounted for the difference in disease severity? The researchers decided to sequence the entire genome of Ringo, Suflair and one of his severely affected male puppies. (DMD is an X-linked disorder that only affects males.) And then compare.

“We asked, ‘what did the father pass to the escaper that made him able to escape the disease?’” says Vieira.

That led them to a sequence of DNA that functions as a promoter, turning Jagged1 on. As a result, the escaper dogs, which carried a slightly different sequence in that location, expressed Jagged1 at twice the rate of the affected dogs.

Zebrafish drug screens

To confirm that Jagged1 explained the difference in disease severity, Vieira and Kunkel moved to zebrafish engineered to carry the DMD mutation. These fish have muscles that are clearly broken and disorganized, and they are visibly weak.

“They’re basically immobile; if you touch them, they only move a little,” says Kunkel. “It’s completely compromised muscle.”

zebrafish muscular dystrophy
DYSTROPHIN, JAGGED1 AND MUSCLE: Above, a wild type zebrafish (top image) and a zebrafish lacking functional dystrophin protein. Below, zebrafish lacking the dystrophin protein (far left) show broken, disorganized muscles, but those that also express extra Jagged1 (four middle images) show normal muscle structure similar to fish with intact dystrophin (the two right-hand images).

The zebrafish at far left lack the dystrophin protein and show broken, disorganized muscles  (especially evident with the red stain). In the four middle images, zebrafish lacking dystrophin but expressing extra jagged1a or jagged1b show normal muscle structure similar to fish with intact dystrophin (the two right-hand images).

But when Vieira and Kunkel artificially stimulated Jagged1 expression, they found that the fish, despite being dystrophin-deficient, had normal-appearing muscles and swam normally.

The findings make Jagged1 a new potential target for therapies aimed at improving muscle function, says Kunkel.

“We’re trying to mimic the effect of the promoter and trying to upregulate Jagged1 in fish and mice, using small molecules,” he says. “Zebrafish are permeable to small molecules and have a muscle phenotype that you can score.”

The decade of therapy

Kunkel, who first identified dystrophin in 1987, notes that other therapies for DMD are in the clinical pipeline. One approach tricks the cellular machinery into making dystrophin, making DMD milder; another increases levels of utrophin, a protein much like dystrophin that could compensate for its absence. Still other approaches seek to address problems caused by dystrophin loss, such as reversing impaired production of nitric oxide to improve blood flow.

Jagged1 upregulation is just another avenue for therapy that needs to be pursued,” Kunkel says. “The different approaches work on different systems and are going to be complementary.”

The research published in Cell was supported by the NIH’s National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR064300), the Duchenne Foundation, the Muscular Dystrophy Foundation, FAPESP-CEPID, CNPq, INCT, AACD, FID and the Bernard F. and Alva B. Gimbel Foundation.

Here’s some video of Ringo and Suflair: