Limb deficiencies are among the most common and disabling of all congenital anomalies. Many conditions, such as tibial hemimelia, involve the femur and anterior structures such as the tibia (Fig.1) and the great toe. The genetic root of most of these anomalies is still unknown and the embryologic basis by which the affected limb elements develop normally is scarcely understood.

Dr. Hopyan has developed a mouse model for congenital femoral and tibial deficiency that he believes is unprecedented and represents a potential breakthrough that will allow the subsequent identification of human mutations that cause femoral and tibial deficiency.

“We identified a previously unrecognized population of progenitor cells that generate the proximal and anterior skeletal elements of the limb. We also learned that these cells are specified early, prior to limb bud outgrowth,” he said.

“Our hope is that basic science will enable us to achieve quantum leaps for the care of patients with limb deficiency. With clinical investigation, the best we can do is optimize existing therapies. But we can’t make big strides unless we have new biologic information. The lab is my place to conceive management strategies for the future,” said Dr. Hopyan.

Tracking limb development
A substantial number of human congenital limb deficiency syndromes involve skeletal elements relevant to Dr. Hopyan’s research, namely the femur, tibia, and digits one and two. Many of these deficiencies are not inherited and it is not known how they develop.

Now, Dr. Hopyan and his team have re-created the condition in a mouse model by generating both mutant and reporter genes. Reporter genes have easily measurable phenotypes, so they can be used to follow the effects of a cascade of signaling events on gene expression inside cells. This allows the team to mutate some cells but not others so that they can observe the function of key genes in the mutant limb. They genetically label cells and, with time-lapsed live imaging of mouse embryos, follow the behavior of tissues to determine how the mutated genes regulate skeletal pattern that is deficient in the embryo.

“Having a mouse model of a human condition is extremely powerful,” Dr. Hopyan explained, “especially when you know the genes that are mutated. That puts us in a unique position to dissect the basis for that condition—not only at the basic genetic level but also the molecular and cellular mechanisms in a mammalian model that is very relevant to the human condition.”

Dr. Hopyan believes this work is relevant to understanding how progenitor cells organize themselves to pattern the limb. “Perhaps someday we will be able to make a limb,” he said. “First, though, we must understand how the embryo does it. Although we might not be able to reverse structural deficiencies during development, we might ultimately be able to devise strategies to recapitulate embryonic development of deficient structures.

“That’s a goal for the very long term—beyond my lifetime, but we need to start somewhere,” he continued. “If we don’t have the information we need to make the advances we want, we have to go get it and we do that through research. We mustn’t be disappointed that quantum leaps could be a long way off.”

A scientist first
Dr. Hopyan’s interest in science preceded his interest in medicine. But his involvement in both is motivated by the desire to leverage care. As a physician, he can care for a finite number of patients; “in science, we can contribute to positively affecting a very large number of people in a profound manner that is well beyond the scope of my limited tools and career span as a surgeon,” he said.

OREF, Dr. Hopyan said, is aware of the impact research can have. “More than most general science funding agencies, OREF appreciates the magnitude of orthopaedic problems and the degree of morbidity in North America and around the world attributable to traumatic and congenital limb deficiencies.”

For Dr. Hopyan, the OREF grant filled a real need. “This organization provides an essential step up for early career researchers to build upon new ideas until they are ready to compete with established scientists for national grants from agencies such as the National Institutes of Health and Canadian Institutes of Health Research.

“Science can be very creative and motivating,” he continued. When we meet in the lab to discuss what’s happening with our work, there’s often a sense of awe when we realize we’ve stumbled onto something that no one else has seen or recognized before. We then compose these moments into a story for our research papers.

“It’s like being an explorer and seeing something for the first time,” he said. “When we get to share it with others, those are big highs. We want to contribute to understanding limb development well enough to facilitate future quantum leaps in clinical care.”

Catherine Rategan is a contributing writer for OREF. She can be reached at communications@oref.org