The toddler's eyes were frozen in an uncontrollable stare at the floor and his eyelids drooped over the whites of his eyes. To look up at neurology resident Elizabeth Engle, he had to tilt his head way back. A conversation with the boy's mother revealed that this defect ran in the family, but they knew nothing about what caused it—or even if it had a name.
Engle didn't know either, so she referred the boy for a battery of neurology and ophthalmology tests. A short note back from the consulting ophthalmologist stung: "Why are you putting the child through all these tests? He has a well-described syndrome that's been in the ophthalmological literature since the 1800s. Congenital fibrosis syndrome."
Embarrassed that she hadn't recognized the syndrome, Engle—now an HHMI patient-oriented researcher—went straight to the literature. True, the disease was well described, but it wasn't well understood. She was determined to look at it with fresh eyes.
Engle saw the case as a chance to put fledgling genetic technologies to the test and track down the gene that caused the disorder. She didn't consider it a problem that she had no idea how to do the required experiments, or that she knew nothing about the eye disorder; she viewed it as a learning opportunity. That mindset has characterized Engle's career.
"Elizabeth had a fire in her belly to really do research," says Alan Beggs, an early mentor of Engle's.
Explore the molecular details of one eye disorder.
That 1992 encounter with the young boy at Children's Hospital Boston led pediatrician and neurologist Engle to become a scientist who follows the trail of her research wherever it takes her: genetics, developmental biology, neuroscience, cell signaling. She's discovered how a class of rare eye disorders, including the one affecting the boy, are caused by mishaps in the development of the nervous system, and she's linked specific gene mutations to seven of the disorders.
At each fork in the road, she chooses the gutsiest way forward, the path she hasn't traveled before. The course of her career has been as complex and circuitous as that of the developing neurons she studies. Now, she's closer than ever to her final destination: complete understanding of the disorders.
The Brain's Intricacies
Science wasn't always forefront in Engle's mind. She grew up in Columbus, Ohio, devoting her spare time to playing the cello. Then, in junior high school, she met a teacher who took students on annual treks to the Bahamas to study marine biology.
"My parents are both academics, and I learned early on that they would support my interests if they viewed them as educational," says Engle, "and I thought this was the perfect way to go on a cool trip." She got more than just a tan, it turned out. She got hooked on marine biology and returned to the Bahamas throughout high school. "I could spend hours snorkeling over the Andros Barrier Reef watching fish dart among coral, or floating in a mangrove ecosystem watching crabs move about," she says.
But Engle's interests evolved as a freshman at Vermont's Middlebury College when she enrolled in a genetics course because she couldn't get into her desired geology class. She found herself addicted to the testable hypotheses that genetics presented. "I loved to ask questions when I knew there was a real foundation for an answer and an organized way of getting there," she says. As her interests in the cello and marine biology faded, Engle got a job as an undergraduate research assistant in a bacterial genetics lab at Middlebury and then debated her next step. She was interested in both research and patient care. She chose to go to medical school, figuring that would leave both doors open.
At the Johns Hopkins University School of Medicine, Engle's first anatomy class sparked another new interest: the brain. "I hadn't thought much about how the brain was put together prior to med school," she says. "To suddenly look at this puzzle that is the brain, with its precise tracts and complicated anatomy, was so fascinating."
With her overflowing interests, Engle chose to do two residencies—one in pediatrics at Hopkins and a second in neurology at Children's Hospital Boston. She spent a gap year between them in a fellowship in neuropathology—that is, autopsies of the brain—at Boston's Massachusetts General Hospital.
"I had this need to know what a diseased organ looks like, feels like, smells like," Engle says. "To really get to know what a disease is, I felt it was necessary to look at it grossly and microscopically, and to put my hands on it." Though she's no longer elbow deep in body parts, Engle has an innate need to understand what makes the body tick.
Under the tutelage of E.P. Richardson, whom Engle calls "one of the great fathers of neuropathology," she learned her way around a pathology lab. She spent her days looking at brains, cutting them up, preparing samples, and looking at them under a two-headed microscope with Richardson. She grew to know, and love, the intricacies of the brain. It's an experience that few neurologists get now, Engle says, since MRI technology has become the go-to way to understand neuroanatomy and diagnose neurological diseases.
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Three years later, in the last year of her neurology residency, Engle was forging ahead with her plan to learn genetics by studying the toddler's disorder, specifically called congenital fibrosis of the extraocular muscles (CFEOM) type 1. For help, she approached Beggs—a colleague at Children's Hospital studying muscular dystrophy with former HHMI investigator Louis Kunkel.
"She literally walked up to me in the quad one day with this proposal," says Beggs. "I was quite taken aback."
In muscular dystrophy, the eye muscles are spared from the progressive muscle weakening that affects the rest of the body. So Engle presented her idea to study CFEOM type 1 to Beggs and Kunkel as a project that could answer some of their questions about the uniqueness of the eye muscles. They agreed to take her on.
We did what I felt would be most exciting and meaningful in the long haul. Just follow the trail wherever it leads us.
Engle and Beggs drove to the toddler's home in New Hampshire and spent an afternoon sitting in the family's kitchen meeting relatives affected by the disorder. Engle collected blood samples, Beggs labeled the tubes, and they both observed the signs of CFEOM. They would do this many more times over the next three years. As she'd hoped to do, Engle learned the ins and outs of running a genetics study—everything from enrolling patients to using the novel technologies of the 1990s to zero in on the genetic mistake causing a disorder.
"The time that we worked together was marked by a very steep learning curve for Elizabeth," says Beggs. "And she learned things much faster than anyone I've ever worked with."
Only a few years later, in 1994, Engle's search for the gene causing CFEOM type 1 led her to the center of chromosome 12—one of the 23 chromosome pairs in which every human cell packages its genetic information. Frustratingly, at that time the centers of chromosomes were notoriously hard to decode, packed with the most complicated and repetitive strings of DNA. But the Human Genome Project was under way, and Engle knew it wouldn't be long until other scientists published a complete genetic map of chromosome 12. So she made a calculated move: she stepped back to wait for the map of the chromosome before finishing her gene hunt.
Her lab spent the next several years building up what Engle calls their "treasure chest" of rare eye disorders—finding families, defining their disorder, and then determining which ones fell into groups. "I admit I am a tenacious individual," says Engle. "I would travel all over the country finding families. I would wait in someone's driveway until midnight when they got home so I could enroll them in our study, because maybe they'd be the person that would get us that much closer to a gene."
By 2003, Engle had pinpointed the mutations that cause CFEOM type 1, as well as CFEOM type 2, which she'd discovered in families in the Middle East. Both mutated genes suggested that the root cause of CFEOM disorders was in the development of neurons, not in the stiffening of the eye muscles (as was previously thought and suggested by the "fibrosis" in CFEOM). Engle was intrigued by exactly how the genetic mutations caused the developmental problems. But she knew little about this area of biology.
Her lab had the choice of continuing to identify genetic causes of eye disorders, and then pass the genes along to other groups to study, or delving into the molecular details of the disorders themselves. She chose the harder path, heading straight to developmental biology textbooks. "We did what I felt would be most exciting and meaningful in the long haul," Engle says, "just follow the trail wherever it leads us."
That same year a graduate student in neurobiology approached Engle, interested in working in her lab. Max Tischfield arrived at just the right time and was a key player in the lab's evolution over the next seven years, bringing his own expertise in developmental neuroscience, and basic research, to the then largely clinically oriented lab. He led the effort to study the latest set of gene mutations that the Engle lab had discovered—work that drew the lab into studying the role of microtubules in nervous system development.
Tischfield, now a postdoctoral fellow in the lab of HHMI investigator Jeremy Nathans at Hopkins, says, "I'm very proud of where Elizabeth's lab is today. When I joined, the lab was very much clinicians, trying to classify these diseases. Now, there are experimental neuroscientists, cell biologists, geneticists. That was the natural transition that the lab was headed for, and I'm very glad I got to be there for that."
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Correcting Genetic Mistakes
Neurons are born amidst a concoction of chemical signals in a developing embryo's brain. Each cell's location in the brain is genetically programmed. Moreover, each neuron must stretch its long axon out to a specific location in the body—be it a big toe, a liver, or an eye muscle. To get there, each neuron follows chemical road signs, which interact with unique sets of surface proteins on each axon's tip. For the axon to lengthen, stiff structural filaments—called microtubules—arrange themselves in the right direction inside the neuron. Microtubules also become the highways that carry materials and messages between the axon and the neuron's control center, back in the brain.
Engle's lab has discovered that when it comes to the neurons that lead to the eye muscles, mistakes at any step of this process can lead to disorders. Some disorders are caused by a mutation in genes that give these neurons their identities. Others develop from mistakes in the proteins on the tips of axons. In these cases, the axons veer off in the wrong direction and never reach the eye. Microtubules that don't form properly cause another set of disorders, and malfunctioning molecular motors that carry cargo along the microtubules cause yet another, thwarting the relay of messages from the brain. Engle's team has linked mutations to seven complex eye movement disorders, including CFEOM types 1, 2, and 3; horizontal gaze palsy; and Duane's retraction syndrome.
"Wiring the brain to the periphery requires remarkable and complex genetic programs," Engle says, "and as you might imagine, these developmental programs can go wrong." Humans likely have variability in the exact projections of their axons to muscles, she hypothesizes, but in most cases, these errors probably go undetected. "A crooked smile is viewed as charming rather than debilitating. A small error in wiring to the huge quadriceps muscle may make you less of an athlete, but wouldn't be very noticeable."
But eye movements must be precisely controlled. Your left and right eye must point in the same direction, focus on the same object, and move at the same time for ideal vision. We use our eyes to communicate, express feelings, and explore our world. Complex eye movement disorders turn out to be sensitive indicators of wiring gone wrong.
Back to the Patients
Since 2008, Engle's been running a monthly clinic with David Hunter, chief of ophthalmology at Children's Hospital and vice chair of ophthalmology at Harvard. Hunter is a specialist in surgeries that, in some cases, can correct complex eye movement disorders. Patients come from around the world, and the two doctors pair up to consult with them—taking on only a couple of patients in an afternoon so they have ample time to explore every aspect of each case. Using the genetic information that Engle has uncovered gives Hunter a better idea of what symptoms to ask the patients about and what to expect when he goes into surgery.
Cases of the disorders vary greatly in severity. Some disorders affect both eyes and are often accompanied by other developmental disabilities; others rarely affect anything more than one eye. Some cause drastic paralysis of particular eye muscles, while others can be corrected with glasses or patches.
"We're at the stage now where we see these patients knowing what the mutation is, and so when I do surgery I at least know that going in," says Hunter. He can often tweak the placement of an eye muscle so that the eye's default position is forward, rather than a fixed gaze downward or to the side.
"A lot of people didn't think it was a good idea for her to study such rare disorders," Hunter says about Engle. "It's just a testament to her persistence that it didn't matter to her what people said. It wasn't about trying to make a big discovery, it was about trying to help these patients."
But bringing the science back to the patients is especially tricky in the case of CFEOM, Engle is the first to admit. "Translating genetic research back to the patient is often challenging," she says, "but for these complex eye movement disorders it is particularly so. These neurons are born and extend their axons at around 4 to 6 weeks of human gestation; the developmental errors occur before a woman may know she's pregnant."
Engle and Hunter's clinic offers diagnostics and genetic counseling for those more severely affected who want to prevent the disorder in their children. But scientists aren't yet at the point where they can interfere with early developmental processes in the brain once the disorder is inherited.
There are still plenty of questions about CFEOM and other complex eye movement disorders that Engle hopes to answer. Many of the genes that are mutated in these disorders are in every neuron in the brain, and in some cases, other organs as well. So why aren't other parts of the brain and body affected? And though Engle has identified the gist of how some of the mutated genes cause a disorder, there are still molecular caveats to work out. With a powerful new microscope that can zoom down to single molecules, for example, she's hoping to understand the effect that microtubule mutations have in the neurons leading to the eye. Is the microtubule too stiff or not stiff enough? Does cargo fall off the microtubules as it tries to move along? Does it stall in place? Where these questions will lead her next is anyone's guess.
Engle is incredibly devoted to her work and very intense—in a good way—says Beggs, and if anyone can answer these questions, she can.
"If reviewers of one of her papers come back and request some additional work that will take six months, a lot of authors might just try submitting it to another journal," says Beggs. "But she'll start whole new collaborations and learn new methods to address one of these questions. She's so precise in doing the best science that she can."