A genetic disorder caused by silencing of a gene on the X chromosome, fragile X syndrome affects about one child in 5,000, and is more common and more severe in boys. It often causes mild to moderate intellectual disabilities as well as behavioral and learning challenges.
The gene responsible for fragile X syndrome, the most common inherited form of intellectual disability, was identified more than 25 years ago. Emory genetics chair Stephen Warren played a major role in achieving that milestone. His work led to insights into the molecular details of learning and memory, and nationwide clinical trials — which have a more complicated story.
Treating the molecular basis of a neurodevelopmental disorder, instead of simply addressing symptoms, is a lofty goal – one that remains unfulfilled. Now a new study, supported by the National Institute of Neurological Disorders and Stroke, is reviving a pharmacological strategy that Warren had a hand in developing.
“This is a very well thought out approach to studying changes in language and learning in children who are difficult to test,” says Amy Talboy, medical director of Emory’s Down Syndrome and Fragile X clinics, who is an investigator in the NINDS study. “It could change how we conduct these types of studies in the future.”
Flip the classroom
Working with Warren, Talboy and others at Emory, medical illustrator Michael Konomos has assembled an iBook – available free through Apple iTunes – introducing medical students to the science of fragile X and its history using multimedia. This summer, the iBook won an “Award of Merit” from the Association of Medical Illustrators in the Interactive Textbook category. It contains video interviews with Warren and Talboy, along with animated depictions of how the molecules involved in fragile X syndrome lead to differences in learning and behavior.
“Genetics and neuroscience are often taught to medical students by professors leaning heavily on projected slides in the classroom, giving little insight into the vast molecular world that exists within the human body,” Konomos says. “The iBook was designed to depict basic science in a way that would interest medical students, not only teaching them about the particular subject of Fragile X, but awakening their own interest in engaging in fundamental science research themselves.”
Medical educators Gordon Churchward and Kathryn Garber brought the iBook into the genetics curriculum for first-year students in the fall of 2017, and are planning on using it again this fall. It’s part of an effort to use non-lecture-based (“flipped classroom”) teaching strategies.
“What Michael has put together is an amazing platform for providing information. I think the students found it refreshing,” Garber says. “What stood out to them especially was the material from Amy Talboy. That goes so much farther than a bullet point list of symptoms. She has a lot of experience with individuals with fragile X – and it’s not always so easy to bring them into an intimidating classroom environment.”
Children affected by fragile X syndrome display intellectual disability and delay of milestones such as language. Individual affected childrens’ capacities vary, but fragile X syndrome frequently includes autism-like features, such as impaired social skills, gaze aversion/social anxiety, hyperactivity and repetitive behaviors. The iBook uses artists’ depictions to portray the syndrome’s characteristic head shape and facial features.
In her interview, Talboy outlines how building rapport and working with affected families are important: She says: “We don’t make the child with fragile X the center of attention immediately. If they don’t want to come into the room, we don’t make them… You really have to take quite a bit of time. This is not a 15 minute appointment. It may not even be the first visit.”
A genetic anomaly
In his sections, Warren describes the “Sherman paradox,” named after colleague Stephanie Sherman, who observed that the number of affected males in fragile X families tended to increase generation by generation. For leaders in the field at the time, this well-documented phenomenon was confounding, he says.
The anomaly was explained by an expanded triplet repeat (CGG). With each generation, the number of repeats tends to rise. So if a mother has a dozen repeats, her child may have 13 or 14. At some point, the repeats reach a tipping point—mothers who have 55 or more often have children with hundreds of repeats—which caused the anomaly that Sherman noticed
Carriers of the pre-mutation can have their own challenges to deal with: fragile X-associated tremor/ataxia syndrome and fragile X-associated primary ovarian insufficiency.
“What we found out about fragile X syndrome was a fundamental change in the way we think about genetics,” Warren says in his iBook interview. “Ultimately, we found that the cause of the mutation was a long expansion of a simple repeat. That had never been discovered before in any genetically studied organism, let alone human disease.”
The finding was instructive for researchers studying other disorders such as Huntington’s disease and amyotrophic lateral sclerosis (ALS), which have also been linked to triplet repeats, he adds.
Warren provides glimpses of how the genetics emerged, in the days before the Human Genome Project. He notes how researchers used homegrown pulsed-field gel electrophoresis equipment, which were not commercially available at the time, for analyzing especially large DNA fragments.
To explain the origins of the genetic mutation, Konomos turned to molecular animation, a specialty that is becoming increasingly sophisticated. Depicting how cells’ DNA copying machinery can slip took weeks to get right, he says. Computer models of relevant proteins from the Protein Data Bank were brought into Cinema 4D, a software used for medical and commercial animation. Konomos taught himself the software along with a molecular plug-in, EPMV.
Showing the effects of FMR1’s silence on neurons and thus, how fragile X syndrome perturbs learning and memory, was even more of a challenge. One scene, a section depicting the alterations of proteins inside the dendritic spine of a neuron, was particularly difficult to pull off.
“How do you accurately portray how molecules interact at the synapse, or in the nucleus?” he says. “Molecules move by random motion. They don’t have brains. They aren’t on a mission. They are just floating and to some degree bouncing around…I had to tell these little guys to run around, but not TOO much. Basically, it was like have a bunch of dogs running around inside a fenced-in little dog park, and trying to direct the dogs to go in certain ways at certain times.”
Twists and turns
The iBook doesn’t dive into this as much, but Warren’s work on fragile X led eventually led to clinical trials, which have had their own twists and turns. A wave of enthusiasm for fragile X clinical trials resulted in disappointment. Emory experts say that along the way, investigators have learned a great deal about how to design clinical trials and how to measure a child’s progress.
“Designing clinical trials for intellectual disability is just tough,” says Warren. “It’s not like measuring blood pressure.”
A decade ago, Warren’s collaboration with Mark Bear at Brown/MIT, which started through a chance meeting at a Howard Hughes Medical Institute conference, built an influential idea of how the neuronal basis of fragile X syndrome might be treatable: the mGluR5 theory.
FMRP, the protein encoded by the FMR1 gene, restricts protein synthesis in neurons. It ensures that changes at critical neuronal connections take place in response to outside signals. In FMRP’s absence, protein synthesis occurs indiscriminately. The mGluR5 theory proposed that inhibiting a particular neuronal receptor (mGluR5), whose role is to excite protein synthesis, could compensate for the problem.
The idea was tested in mice and then in clinical trials, in which drugs targeting mGluR5 glutamate receptors were tested in adolescents and adults. In humans, mGluR5 drugs did not show clear benefits; re-evaluation suggests the choice of outcome measures, drug tolerance, and the ages of study participants may have played a role.
“Fragile X syndrome has a genetic origin, but there is great variability,” says Ami Klin, director of Marcus Autism Center, the country’s largest center for clinical care of autism. “On top of that, a child’s environment and upbringing are overlaid upon the genetics. Disability accrues over time.”
That means that focusing on children at an earlier age and a narrower age range, although more difficult to implement, could be wiser, Klin says. In addition, initial studies relied on questionnaires filled out by parents, which didn’t provide that much fine detail. Tracking more sophisticated behavioral measures will be key to evaluating a child’s progress, he says.
Recently, one of mGluR5 clinical studies has been revived in younger children (ages 3 to 6), supported by NINDS. The clinical trial looks at the effect of an intensive language intervention aimed at improving communication skills, paired with either a mGluR5 drug called AFQ056 or placebo. Talboy’s center at Emory is one of the sites, starting in the fall of 2018.
“We are really evaluating the child’s ability to learn language,” Talboy says. “This is a critical period, when a child’s brain is still developing. The time frame is actually longer than in previous studies.”
Another pharmacological strategy for the treatment of fragile X syndrome gained support from the Warren lab’s work with Drosophila fruit flies. His team found that flies with mutations in their version of the FMR1 gene were especially sensitive to having glutamate in their diet. (This was a serendipitous discovery, because lab-prepared food was superior to commercial food.) GABA, a key mover in an inhibitory pathway in the brain, could keep the flies alive and rescue behavioral defects.
A drug that acts upon one type of GABA receptors, arbaclofen, made its way to clinical trials in adults, and in children as young as 5. Emory Medicine had a feature on the family of a boy who was participating in the arbaclofen study. “Samuel is a very happy, easy-going, joyful boy,” says his mother. He loves trains, baseball, and riding his Big Wheel.
Unfortunately, these studies ran aground as well. Still, clinical trials, like the one Samuel participated in, do help researchers understand the best ways to measure children’s cognition and behavior. The Simons Foundation, which supports autism-related research, has acquired the rights to arbaclofen, with an eye on a future re-do.
Cell biology chair Gary Bassell, collaborating with Shannon Gourley at Yerkes and his former postdoc Christina Gross at Cincinnati Children’s, has been developing an alternative drug strategy. It is also aimed at taming indiscriminate protein synthesis, but with a different target: a particular form of the enzyme PI3 kinase. In a recent paper, the three labs showed that an inhibitor of p110beta PI3 kinase relieved symptomatic behaviors, such as impaired social interactions and inflexible decision making, in the mouse model of fragile X syndrome.
“We can’t use mouse behavioral tests alone to understand a drug’s effects,” Bassell says. “It is critical to have molecular, cellular and neurophysiological phenotypes, which we and others do.” (See two related items from Spectrum News on this topic. Elizabeth Berry-Kravis + mouse models, more recent roundup including Bassell’s work)
For his part, Warren thinks that drug strategies aimed at replacing FMRP’s regulation of protein synthesis target just one aspect of its function. His lab showed that the tendency of children with fragile X syndrome to develop seizures is independent of regulating protein synthesis.
“The most effective strategy is likely to be reactivating the fragile X gene,” he says, adding that this could be possible through epigenetic approaches or even gene editing. “After all, the gene itself is intact. There’s nothing wrong with it, except that it’s turned off. That’s what I’d put my money on.”
One of the reasons big pharmaceutical companies were interested in fragile X was because it was perceived as a gateway to the more common (and more heterogenous) diagnosis of autism. Now, more in the field are emphasizing the fine distinctions. Warren was quoted in a 2014 story in the Simons Foundation’s Spectrum News:
Children with autism and those with fragile X syndrome both shy away from social contact, have trouble making friends and avert their gaze when people look at them. But children with fragile X syndrome often sneak a peek when the other person turns his back, researchers say. Children with autism, in contrast, seem mostly uninterested in social interactions.
“Children with fragile X syndrome all have very severe social anxiety that plays a big role in the perception that they have autism,” says Stephen Warren, professor of human genetics at Emory University School of Medicine in Atlanta. “They are actually interested in their environment; they are just very shy and anxious about it.”