Insight

Cracking the Coloboma Code

Research calls for lab and clinic collaboration

By Allyson T. Collins, M.S.
NEI Science Writer/Editor

Brian Brooks, M.D., Ph.D., Chief, NEI Unit on Pediatric, Developmental and Genetic Ophthalmology Research

In the late 1990s, Brian Brooks, M.D., Ph.D., trained as a pediatric ophthalmology fellow at the University of Michigan. One day, a couple that were expecting their second child arrived for an appointment in the clinic.

The parents explained to Brooks that their first child had been diagnosed with coloboma, a congenital eye condition that researchers estimate causes up to 10 percent of childhood blindness. They knew that their next child could be born with the condition, so they asked Brooks if he could figure out the chances of that happening.

"I didn't know the answer," he says. He turned to textbooks and research papers, only to find that no one had the answer.

"I thought this was an opportunity to fill a void," Brooks says. After finishing additional training in genetics at the National Human Genome Research Institute, he came to the National Eye Institute (NEI) in 2005 to establish the Unit on Pediatric, Developmental and Genetic Ophthalmology Research, in the hopes of finding answers for future families affected by coloboma.

He brought in Prasad Alur, Ph.D., who has a background in biochemistry and research experience in both molecular biology and gene therapy, as the lab manager. While Brooks deals with both clinical and basic research aspects, Alur concentrates on DNA, the molecules that carry the body's genetic code.

"The purpose of our research is to better understand the genetics of coloboma, starting with mouse and zebrafish development, and bringing that information to patients next," Brooks says.

How coloboma happens

A crucial event occurs during the fifth and sixth weeks in which a human embryo develops: the optic fissure closes. In the first few weeks, this gap remains open so blood vessels can nourish the developing eyes. But for the eyes to form properly, the tissue on each side of the opening must eventually come together, like when a zipper seals a suitcase.

When the optic fissure remains unzipped, a person can experience minor to major effects in one or both eyes.

This eye with coloboma shows a keyhole-shaped defect in the iris, which distorts the pupil. (Courtesy of Brian Brooks, M.D., Ph.D., and Mel Palmer, NEI ophthalmic photographer)

Coloboma can appear as a small defect in the front of the eye, giving the pupil a black "keyhole" shape that juts into the normally colorful iris. A person with this abnormal-looking pupil may have perfectly normal vision, Brooks explains.

If the condition extends to the back of the eye--to the light-sensitive retina or the optic nerve that carries visual input to the brain--a person may have limited or no vision.

Defects from coloboma are present at birth. "It is a developmental problem, and once a patient has the condition, it is usually stable and does not progress," says Wadih Zein, M.D., an NEI staff clinician who works with Brooks.

Eye care professionals know what the condition looks like, but they know little about why it occurs, although genetic defects are believed to be responsible for some cases. Brooks has been studying coloboma for the past four years, and has collected genetic and clinical information from about 90 families with the condition.

"Every patient I see is a little bit different, and provides new clues for what might be going on at the molecular, cellular and embryonic levels," Brooks says.

Searching for genetic evidence

Researchers around the world have proposed candidate genes that, when mutated, could be solely responsible for coloboma. However, none of these genes is "high-yield," meaning that out of 100 patients who have the condition, only one might have a certain genetic mutation, Brooks explains.

"We're trying to look at every minor detail in the eye to figure out how coloboma happens and what other factors are associated with it," Dr. Wadih Zein explains.

"Coloboma is much more complex than we originally thought," Alur says. "It could have multiple genes causing it, or there could still be a master gene that we have not hit upon."

In their quest to pinpoint coloboma genes, researchers look to information that they already have about embryo formation and genes involved at different time points. "We use this knowledge to pull out genes that might be related to coloboma," Brooks says.

They need genes that are expressed in the right place at the right time. "At a particular time and in a particular instance, certain genes must be expressed and perform their duties," Alur says.

A former graduate student in Brooks' lab, Jacob Brown, Ph.D., examined tissue removed from the edge of the optic fissure before, during and after it closed in developing mice. He identified 165 possible genes that could be involved in coloboma, but focused on two in particular that were highly active during the closure of the optic fissure, called NLZ1 and NLZ2.

When Brown, Brooks, Alur and their colleagues altered the expression of these genes in zebrafish, the zebrafish showed signs of coloboma. This research, which took several years to complete, was published in the February 2009 Proceedings of the National Academy of Sciences.

"Now, we're moving on to determine the role of these genes in humans," Brooks explains.

But it's not that simple. "In research, we find that the answer to one question leads to several other questions," Brooks says, including how these genes and the proteins they produce interact with other genes and proteins during development.

Seeing both sides

Prasad Alur, Ph.D., lab manager, at work in the NEI Unit on Pediatric, Developmental and Genetic Ophthalmology Research

Some people refer to the eye as a window to the soul. For Brooks, the organ is a window to the past. "When I look at a patient with coloboma, I'm given clues about what went on during their eye development," he says.

This rare window to a wealth of information makes Alur's job easier. He admits that with ophthalmologists nearby, half of his work is already completed. He knows that he should be able to find a genetic mutation in DNA extracted from the blood sample of a patient that Brooks or Zein has diagnosed with coloboma.

"With the clinicians' observations pointing me in the proper direction, my scientific curiosity is properly fed," Alur says. "It's like eating at a buffet. There are so many choices that you can't decide. But if someone tells you that a dish is good, you know what to choose."

Through this laboratory-clinic collaboration, Brooks says his team ultimately hopes to gain a better understanding of the eye's development process.

"We're trying to look at every minor detail in the eye to figure out how coloboma happens and what other factors are associated with it," Zein explains. "Our research has already improved our knowledge of what genes play a role in how the eye is formed. That, in itself, is quite significant."