Montage based on photos of Eduardo CesarFor the last seven years, Ringo, a golden retriever, has intrigued researchers at the Center of Studies on Human Genome of the University of São Paulo (USP), one of the Research, Innovation and Diffusion Centers (Cepid) financed by FAPESP. Ringo has a genetic mutation that impairs the production of dystrophin, an essential protein for maintaining healthy muscles. This should have led the dog to manifest, early on, the classical clinical signs of muscular dystrophy, such as difficulty walking and swallowing, and the dog would probably no longer be alive by now if it had developed the disease. However, Ringo is a healthy, almost normal dog. The genetic mutation only makes him drag his rear legs. Four and a half years ago, Ringo fathered a puppy, Suflair, who inherited the same genetic defect, but has not shown signs of muscular dystrophy. The other puppies from the litter were not as lucky; some died a couple of days after birth, while others developed a severe form of muscular dystrophy.
Ringo and Suflair are exceptions to the rule. This is why they have been the subject of a number of studies – one of which provided evidence of why these dogs’ muscles were not affected by the lack of dystrophin. The dogs seem to carry genes or protective mechanisms that totally or partially neutralize the adverse effects of the mutation that causes the disease. In an experiment conducted in partnership with the laboratory headed by Sergio Verjovski-Almeida, from USP’s Institute of Chemistry, the researchers found that some of the asymptomatic dogs’ genes were less expressed (activated) than those of sick animals. “Our theory is that these genes that are less expressed might provide the dogs with some form of protection and this might be important to help us find a way to deal with this disease,” says geneticist Mayana Zatz, who coordinates the center. “We are breaking a paradigm and showing that the absence of the protein does not always lead to dystrophy.” As they have not yet published a scientific paper on this study, the geneticists from USP prefer not to provide any more details on the location of the possible protector genes.
Mayana’s theory was reinforced when her team became acquainted with the work of veterinarian Diane Shelton, who heads a laboratory at the University of California in San Diego (UCSD). This laboratory specializes in the diagnosis of neuromuscular diseases in pets. Since 2008, the American. researcher has found nine dogs that do not produce dystrophin, yet do not have any of the symptoms of muscular dystrophy. However, these asymptomatic dogs are not golden retrievers. They are all Labrador retrievers and – this is an interesting detail – come from the kennels owned by two breeders in the states of New Hampshire and Massachusetts, neighboring states on the east coast of the United States. The two breeders share the same bitch to breed litters of puppies. “You can’t tell that these dogs have dystrophy when you look at them, “says Diane. “I think that modifying factors played a role which made these dogs clinically normal.”
The news on the asymptomatic Labrador retrievers literally fell into the lap of the Brazilian team. A few months ago, biologist Natássia Vieira, whose doctoral research advisor was Mayana Zatz and who now does research on the use of stem cells in muscular dystrophies, was at a seminar in Boston, where she attended a presentation by Diane Shelton. In addition to telling the story of the American dogs, the veterinarian said, during her presentation, that she would contact Ringo’s ‘mother’, the asymptomatic golden retriever being studied at USP, to send samples of the Labrador retrievers’ DNA. The partnership began right then and there. Diane was immediately introduced to Natássia, who was in the audience, and a few days later the Brazilian researcher got the DNA samples before leaving Boston to return to Brazil.
Comparing the DNA of the golden retriever with that of the Labrador retrievers might speed up the identification of possible molecular mechanisms that provide protection from muscular dystrophy. The strategy was further strengthened when Mayana’s team revealed a small canine secret. “Ringo and Suflair are not pure breeds,” says Natássia. “They have Labrador retriever genes.” One fourth of Ringo’s genome and one eighth of Suflair’s genome come from another breed. Mayana says she was furious when, years ago, she discovered that one of the golden retrievers, a carrier of the mutation in the dystrophin gene, had mated with a Labrador retriever. Today, she is grateful for the fact that she was rewarded with a litter of half-breed dogs. “The cross-breeding may have prevented Ringo and Suflair from having the disease.”
The search for possible genes that could make up for the absence of dystrophin production and avoid the manifestation of the disease’s symptoms led the team from USP to set up an international partnership with one of the best known experts in the study of genetic bases for muscular dystrophies: Louis Kunkel, a Harvard Medical School professor. In 1987, Kunkel discovered the cause of Duchenne muscular dystrophy: he identified the dystrophin gene in the X chromosome and noticed that when the dystrophin gene is the target of mutations, it no longer produces the respective protein. “If we discover that another gene or other genes, besides dystrophin, can really modify the characteristics of the disease in dogs, these genes and their proteins could be prime targets for therapies in human beings,” Kunkel states.
While working at the laboratory headed by the American researcher, Natássia is using the zebra fish, a fish used as a model organism for the study of diseases, to discover whether the genes that might provide protection against muscular dystrophy identified in Ringo and Suflair mitigate or even prevent the symptoms of the disease. “Our idea is to make the alleged protector genes be expressed in fish with dystrophy in the same manner as in the asymptomatic dogs and see the clinical repercussions of this approach,” says Natássia. If this strategy is successful, the researchers will probably have made a major discovery. If it is unsuccessful, then further studies will have to be conducted on Ringo, Suflair and their asymptomatic Labrador retriever ‘cousins’. However, the news that dystrophin-free muscles can be functional is very exciting indeed.
Center for Studies of the Human Genome (nº 1998/14254-2); Type Research, Innovation, and Diffusion Center (Cepid); Coordinator Mayana Zatz – Biosciences Institute of USP; Investment (annual) R$1,600,000.00 and US$980,000.00 (FAPESP)