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Researchers from Seattle Children’s and Novartis shed light on intractable epilepsy.
Electrical signals pulse through the gray matter of your brain,
allowing you to read and understand this sentence. The cerebral
cortex—home to your gray matter—is packed with more than 20 billion
neurons, which are organized into circuits.1 Collectively, these circuits are the seat of human cognition. And the results can be dire when they don’t form properly.
Take children with a disease called focal cortical dysplasia (FCD).
Born with an enlarged, disorganized area of the cortex, these patients
often experience seizures, brainstorms of uncontrolled electrical
activity that can lead to developmental delays and disabilities. In
fact, FCD is the most common cause of intractable epilepsy in children.2
For years, researchers speculated about the underlying mechanisms of
the disease. Could a virus be triggering the brain overgrowth? Or maybe
the tissue had been bumped and bruised during pregnancy?
Scientists now believe that many cases of FCD have genetic roots. Collaborators from Seattle Children’s Research Institute (link is external),
the Novartis Institutes for BioMedical Research and other organizations
recently traced four cases of FCD to genetic mutations, publishing
their findings online in JAMA Neurology (link is external).
Specifically, the team identified mutations in a molecular pathway
called mTOR, which plays an essential role in regulating cell growth.
The researchers also found MTOR mutations in six patients with more
widespread, diffuse brain overgrowth. The discovery bolsters a growing body of evidence (link is external) that such diseases can be genetic and suggests new treatment approaches.
“We found that there are genetic changes in the brain tissue of these
patients and showed that the changes are related to the structural
abnormalities that occur in the brain,” says first author Ghayda Mirzaa,
a physician-scientist at Seattle Children’s Research Institute. “Now we
have a chance to test molecularly-targeted therapies in epilepsy.”
Clues in patient tissue
Mirzaa’s colleague William Dobyns (link is external),
last author on the new study, began building a registry of patients
with FCD and other brain overgrowth disorders in 1990. The goal was to
learn more about them and identify new therapeutic approaches.
By 2012, the research team at Seattle Children’s Hospital, in
collaboration with the neurosurgical team, led by Jeff Ojemann, had
gathered tantalizing clues by studying brain tissue from the patients.
Some children with the disorders undergo epilepsy surgery, a treatment
of last resort and a source of invaluable samples. If patients fail to
respond to anti-epilepsy medication, then neurosurgeons may remove the
portion of the cortex that’s generating abnormal electrical activity in
an attempt to block further seizures. This precious tissue gives
scientists a window into the disorders.
Biochemical tests indicated that the mTOR pathway was overactive in many of the samples. DNA sequencing revealed mutations (link is external)
in key components of the pathway, but only in patients with diffuse
brain overgrowth. The mutations didn’t show up in any patients with FCD.
Mirzaa and Dobyns, who are also clinical geneticists at Seattle
Children’s Hospital, suspected that the mutations were simply hiding due
to a quirk of biology. High school students learn that an individual’s
DNA is determined when sperm and egg meet. But this is an
oversimplification. While DNA is generally replicated faithfully as
cells divide in a developing embryo, there are some exceptions. As a
result, two or more populations of cells with different DNA can exist in
the same organism, a phenomenon known as mosaicism.
The Seattle Children’s researchers wondered if there was a small
population of neurons with mTOR mutations in FCD patients. Perhaps the
population was so small that the mutations weren’t registering with
standard DNA sequencing techniques.
Uncovering hidden mutations
Luckily, Wendy Winckler’s next-generation sequencing group in Oncology at Novartis had the tools to test the hypothesis.
“Being a cancer sequencing lab, we specialize in finding mutations
that only occur in a small fraction of cells,” says Winckler. “A tumor
is a mix of normal cells, immune cells and cancer cells, so we have to
be able to detect low-level mutations in samples.”
Zeroing in on protein-coding genes, her group performed deep
sequencing on the samples, reading tens of thousands of cells in each
one. The team also sequenced tissue from the patients’ parents and from
the periphery of the patients (blood, saliva or skin) so that they had a
basis for comparison. Bioinformatician Katie Campbell then analyzed the
data.
“We’ve tuned our software to catch mutations that occur in less than 5
percent of the cells,” explains Campbell. “We also have experience
finding mTOR mutations because they’re relatively common in tumors,
given that the pathway regulates cell growth.”
Campbell analyzed samples from eight patients with FCD and their
parents. She identified mTOR pathway mutations—including genetic lesions
identical to those seen in cancer patients—at a low level in four of
the FCD patients. Researchers at Seattle Children’s Hospital and the
University of Washington Genome Sciences Center used targeted sequencing
and deep sequencing to screen 93 additional children with unexplained
FCD or diffuse brain overgrowth. They found mTOR pathway mutations in
six of the patients with diffuse brain overgrowth, suggesting that it’s
related to FCD.
In parallel, Novartis scientists within the Developmental & Molecular Pathways and Neuroscience
groups set out to determine exactly how the mutations affect brain
cells. Carleton Goold, Sue Menon and their teams introduced them into
rat neurons, which proceeded to grow very large. The researchers also
tested mTOR pathway activity in the neurons and confirmed that it was
elevated.
The final step was to rescue the swollen cells. When the team applied
an mTOR inhibitor to the mutant neurons, the cells shrank to a healthy
size, pointing toward a potential therapeutic strategy for patients.
“This pathway is extremely well known in the cancer space, but now
it’s coming up as an important target in neuroscience,” says Leon
Murphy, who led the validation effort at Novartis. “I think that we’re
going to see mTOR popping up in other areas as well. It might be
possible to repurpose cancer drugs for these diseases based on
preclinical data and potentially provide patients with more options at
some point.”
Researchers introduced an mTOR mutation identified from focal
cortical dysplasia patients into these rat neurons. The neurons are
enlarged, similar to what is seen in brain tissue from the patients.
Image by Jonathan Biag/Novartis
Pelvig, D. P., Pakkenberg, H., Stark, A K., and Pakkenberg,
B. (2008). Neocortical glial cell numbers in human brains. Neurobiol.
Aging 29, 1754–1762.
Kabat, J. and Krol, P. (2012). Focal cortical dysplasia – review. Pol J Radiol. Apr-Jun; 77(2): 35–43.
