Helping patient families find answers to mysterious neurological diseases
When Bristol Dunlap was born, she was perfectly healthy. But by the time she was three months old, she was failing to reach her milestones and began showing worrisome symptoms.
“She could not lift her head up or sit upright, her body was unusually floppy, and her left eye began turning inward even though her vision was fine,” said her mother Evonia Dunlap. “As my daughter grew older, she was slow to crawl, stand and walk, and had difficulties in chewing, swallowing and talking.”
Bristol was diagnosed with congenital hypotonia, a symptom caused by various neurological and non-neurological conditions, which explained her poor muscle tone throughout her body. While therapy helped her daughter sit, walk and talk, there was one thing Evonia wanted to know: What caused Bristol to develop hypotonia?
After seeing numerous specialists and undergoing a battery of diagnostic tests and assessments for cerebral palsy, Down syndrome, autism, muscular dystrophy and many others, the results came back negative. Whole exome sequencing — a test that looks for inconsistencies in the genetic code — was inconclusive.
The Dunlaps’ five-year quest for answers finally ended in 2017 at the Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital when investigators made a connection between Bristol’s genes and the genes of other children enrolled in the Undiagnosed Disease Network (UDN). Texas Children’s and Baylor are one of six UDN sites in the country, an NIH-funded program that brings together experts from across the country to help solve mysterious medical conditions by searching for their genetic basis.
The Dunlaps were referred to Baylor College of Medicine’s physician-scientists Dr. Hsia-Tuan Chao, child neurologist and instructor of Pediatrics and Dr. Michael Wangler, geneticist and assistant professor of Human and Molecular Genetics.
Prior to investigating Bristol’s case, Chao had seen a patient named Collette who was born seemingly normal but then began exhibiting mysterious symptoms. At four months, she couldn’t roll over, had difficulty swallowing and holding her head up, and laughed without a smile. Despite extensive diagnostic testing, Collette still had no diagnosis. Upon meeting Bristol, Chao was immediately struck by the similarities between her symptoms and Collette’s.
Through the UDN, Chao and Wangler learned of a seven-year-old boy who exhibited symptoms similar to Bristol’s and Collette’s, and also carried a point mutation in the Early B-Cell Factor 3 (EBF3) gene.
After re-examining Bristol’s exome sequencing results, they found that she carried the same mutation that produces a defective EBF3 protein. Since this protein is a master regulator of hundreds of other genes, even the tiniest alteration in its function could potentially cause widespread damage to the nervous system and muscles.
The team then learned of another little girl at NYU’s Langone Medical Center who had a similar medical history and was found to carry the same EBF3 mutation. Chao embarked on a study of the EBF3 mutations in fruit fly and mammalian research models, and concluded that the point mutation in EBF3 caused the symptoms exhibited by Bristol and the others.
The conclusion was confirmed when Collete’s whole exome sequencing results revealed a slightly different but comparable mutation in EBF3.
“All of a sudden, from this one girl who made such an impact on me, I could finally give all these children and their parents an answer,” Chao said.
A referral center for undiagnosed diseases
Like the Dunlaps, many patient families are referred to Texas Children’s because of the hospital’s renowned expertise and specialization in the diagnosis of diseases that are rarely seen and often unrecognized.
More than 50 percent of UDN patients exhibit neurological symptoms. Texas Children’s Chief of Neurology Dr. Gary Clark is one of the co-leaders of the UDN program at Texas Children’s and Baylor, and works closely with Texas Children’s neurologist Dr. Lisa Emrick in solving these mysterious neurological disorders.
“When a patient is referred to our UDN site, their DNA sample is submitted for sequencing,” said Emrick. “We conduct phenotyping and provide our UDN and NRI partners with the clinical patient data they need to help identify variant genes that may be responsible for a patient’s disease. Before advanced technologies like sequencing, only a small percent of these cases could be diagnosed.”
In addition to state-of-the-art medical imaging, metabolomics and genetic testing including genome sequencing and exome sequencing, clinicians and researchers in the UDN rely on the Model Organism Screening Center (MOSC), where genes are studied in fruit flies to help diagnose patients. The center is led by Dr. Hugo Bellen, professor of Molecular and Human Genetics and Neuroscience and Howard Hughes Medical Institute investigator; Dr. Shinya Yamamoto, NRI investigator and assistant professor at Baylor; and Wangler. This dynamic team uses fruit flies, Drosophila melanogaster, to study new disease candidate genes and variants. They also closely collaborate with researchers at the University of Oregon in generating zebrafish models to study origins of disease.
In the MOSC, researchers combine bioinformatics analysis and experimentation in these ideal organism models to determine whether a specific variant identified in the genome of the patients may be responsible for the disease. “Integration of human genomics and experiments in simple model organisms such as fruit flies and zebrafish greatly facilitates disease diagnosis and mechanistic studies,” said Bellen.
“An exciting technique we developed is a way to humanize a fly gene,” said Yamamoto. “By knocking out the homologous gene in the fly and replacing it with the human gene, we can test the specific variant found in the patient to see how well it performs.”
Since fruit flies share many similar genes with humans, they have become a powerful model organism for the study of genetics. To study human disease in fruits flies, scientists mutate, or disrupt, the same gene that is known or suspected to cause the disease, and then figure out why mutations with this particular gene leads to disease.
Wangler says having such a wealth of experienced neurologists at Texas Children’s is a major asset for neurological research.
“We recently studied a group of patients with mutations in a gene, CACNA1A, which is a calcium channel in neurons,” said Wangler. “Using our fly models, we found different activities in different mutations from the various patients. This led us to see differences between the patients that were not obvious before. It was only because the patients were so extensively evaluated by neurology that the research could make these connections.”
Through collaboration with the UDN — and by leveraging state-of-the-art technologies, robust clinical programs and research in neurology and genetics — Texas Children’s and Baylor continue to make significant strides in helping to accelerate the diagnoses of previously undiagnosed and rare neurological conditions.
For the Dunlap family, this provides a source of encouragement.
“While the journey to find a cure for our daughter’s illness has just begun, we are happy to finally have some answers,” Evonia said. “We are thankful for the pioneering work being done by physicians and researchers at Texas Children’s.”