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Two New Mutations Found in Dogs with Achromatopsia Support Gene Therapy Approach to Curing This Genetic Vision Disorder in Humans and Dogs; Supercomputer Analysis Reveals the Mutations Destabilize CNG Ion Channel Essential to Light Signal Transduction

Cyclic-nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in the CNGA3 and CNGB3 genes, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia. This is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness, and photophobia. The disorder often strikes people early in life, and currently there is no cure for the condition. In human achromatopsia, nearly 100 different mutations have already been identified in the CNGA3 gene alone. Famously, achromatopsia was the subject of a book titled “The Island of the Colorblind,” by neurologist/author Oliver Sacks, M.D. (see image of book jacket). In this work, Dr. Sacks described the surprising prevalence of a particular rare form of the disease (complete achromatopsia) on the remote Pacific atoll of Pingelap. It is believed that the disease on Pingelap can be traced back to a so-called “founder mutation” passed down from an early ruler, who was one of just approximately 20 Pingelap survivors of a 1775 typhoon that hit the atoll. Signifcant inbreeding amongst the Pingelapese is believed to be the reason that much of the island’s small population now suffers from the autosomal recessive disorder, which, in more outbred populations, is relatively rare. One of the most promising avenues for developing a cure for achromatopsi is believed to be gene therapy, but to develop such therapies it is necessary to create animal models of disease that closely replicate the human condition. In a new study, a collaboration between University of Pennsylvania (Penn) and Temple University scientists has identified two naturally occurring genetic mutations in dogs that result in achromatopsia. Having identified these mutations, the scientists then used structural modeling and molecular dynamics on the Titan supercomputer at Oak Ridge National Laboratory in Tennessee and the Stampede supercomputer at the Texas Advanced Computing Center to simulate how the mutations would impact the resulting protein. This sophisticated analysis showed that the mutations destabilized a molecular channel essential to light signal transduction. These findings provide new insights into the molecular cause of this form of blindness and also present new opportunities for conducting preclinical assessments of curative gene therapy for achromatopsia in both dogs and humans.

"Our work in the dogs, in vitro and in silico, shows us the consequences of these mutations in disrupting the function of these crucial channels," said Dr. Karina Guziewicz, senior author on the study and a Senior Research Investigator at Penn's School of Veterinary Medicine.

"Everything we found suggests that gene therapy will be the best approach to treating this disease, and we are looking forward to taking that next step."

The current research began with a German shepherd that was brought to Penn Vet's Ryan Hospital. The owners were worried about their pet’s vision.

"This dog displayed a classical loss of cone vision; it could not see well in daylight, but had no problem in dim light conditions," said Dr. Aguirre, Professor of Medical Genetics and Ophthalmology at Penn Vet.

The Penn Vet researchers wanted to identify the genetic cause, but the dog had none of the "usual suspects," i.e., the known gene mutations responsible for achromatopsia in dogs. To find the new mutation, the scientists looked at five key genes that play a role in phototransduction, i.e., the process by which light signals are transmitted through the eye to the brain.

The researchers found what they were looking for on the CNGA3 gene, which encodes a cyclic-nucleotide-gated (CNG) channel and plays a key role in transducing visual signals. The change was a "missense" mutation, meaning that the mutation results in the production of a different amino acid.

Meanwhile, the scientist heard from colleague Dr. Dixon that he had examined Labrador retrievers with similar symptoms. When the Penn team performed the same genetic analysis on the Labrador retrievers, they found a different mutation on the same part of the same CNGA3 gene where the German shepherd's mutation had been found. Neither mutation had ever been characterized previously in dogs.

"The next step was to take this further and look at the consequences of these particular mutations," Dr. Guziewicz said.

The group had the advantage of using the Titan and Stampede supercomputers, which can simulate models of the atomic structure of proteins and thereby elucidate how the protein might function. That work revealed that both mutations disrupted the function of the CNG channel, making it unstable.

"The computational approach allows us to model, right down to the atomic level, how small changes in protein sequence can have a major impact on signaling," said Dr. MacDermaid, Assistant Professor of Research at Temple's Institute for Computational Molecular Science. "We can then use these insights to help us understand and refine our experimental and clinical work."

The Temple researchers recreated these mutated channels and showed that one resulted in a loss of channel function. Further in vitro experiments showed that the second mutation caused the channels to be routed improperly within the cell.

Penn Vet researchers have had success in treating various forms of blindness in dogs with gene therapy, setting the stage to treat human blindness.

As previously noted, in human achromatopsia, nearly 100 different mutations have been identified in the CNGA3 gene, including the very same one identified in the German shepherd in this study.

The results, therefore, lay the groundwork for designing gene therapy constructs that can target this form of blindness with the same approach in humans.

The described study was published online on September 25, 2015 in the open-access journal PLOS ONE and titled “Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment.”

The article was co-authored by Penn Vet's Emily V. Dutrow and Temple's Naoto Tanaka. Additional co-authors from Penn Vet included Gustavo D. Aguirre, Keiko Miyadera, Shelby L. Reinstein, William R. Crumley, and Margret L. Casal. Temple's team, all from the College of Science and Technology, included Lucie Delemotte, Christopher M. MacDermaid, Michael L. Klein, and Jacqueline C. Tanaka. Christopher J. Dixon of Veterinary Vision in the United Kingdom also contributed.

[Press release] [PLOS ONE article]