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An unusual dog reveals a new approach to muscular dystrophy

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Protective gene overrides dystrophin mutation’s effects

Boston, Mass. (November 12, 2015) – A golden retriever that stayed healthy despite having the gene mutation for Duchenne muscular dystrophy (DMD) has provided a new lead for treating this muscle-wasting disorder, report scientists at Boston Children’s Hospital, the Broad Institute of MIT and Harvard and the University of São Paolo in Brazil.

DMD is one of the most common forms of muscular dystrophy. The study, published online November 12 by the journal Cell, pinpoints a protective gene that boosts muscle regeneration, helping some dogs “escape” the disease’s effects. The Boston Children’s lab of Lou Kunkel, PhD, is now using a zebrafish model of DMD to screen for drugs with the same protective effect.

Natássia Vieira, PhD, a fellow in Kunkel’s lab from the University of São Paolo 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. These dogs were very weak and typically died by 2 years of age, but one dog, Ringo, was able to walk and run normally and lived to the age of 11.

When Vieira joined Kunkel’s lab, they set out to find out how “escaper” dogs have 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, the paper’s co-senior author with Mayana Zatz, DSc, of the University of São Paolo.

Kunkel and Vieira partnered with Kerstin Lindblad-Toh, PhD, at the Broad Institute to conduct the GWAS research. Lindblad-Toh had done genomic studies tracing the evolution of dogs, so was intimately familiar with their genetic complexity. Dogs have highly diverse genomes, and each breed has a different genomic signature.

Combining family linkage analysis and GWAS, the team compared the genomes of two escaper dogs (Ringo and one of his male offspring) and 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 the two escapers 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 and two of his male offspring—the escaper and one severely affected puppy. (DMD is an X-linked disorder that only affects males.)

“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, 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.”

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.”

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. This is the ‘decade of therapy.’”

The study was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH (award number R01AR064300), the Duchenne Foundation, FAPESP-CEPID (# 2013/08028-1), CNPq (#705019/2009), INCT (#2008/578997), AACD, FID (#000663/2014), the Bernard F. and Alva B. Gimbel Foundation and the Muscular Dystrophy Association (MDA352465).

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