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Phenotypic Evaluation Helps Uncover Gene Variant Linked to Childhood Epilepsy

NEW YORK (GenomeWeb) – By folding in a comparison of patients' phenotypes, researchers were able to home in on a de novo gene variant that appears to cause a developmental and epileptic encephalopathy.

Because developmental and epileptic encephalopathies are heterogeneous disorders, an international team of researchers suspected that an approach combining standardized phenotyping and genomic analysis might help elucidate the genetic underpinnings of the condition.

As they reported today in the American Journal of Human Genetics, the researchers analyzed both whole-genome sequences and phenotypic data from more than 300 patients with developmental and epileptic encephalopathies. They uncovered one de novo mutation — affecting AP2M1 — in four patients, all with similar disease symptoms. Through a series of functional analyses, the researchers found that the alteration in this gene affects clathrin-mediated endocytosis and synaptic vesicle recycling, through which the variant could influence disease.

"The main limitation in the past was the lack of large amounts of clinical information in a format that can be analyzed systematically through our informatics approaches," first author Ingo Helbig, a pediatric neurologist at Children's Hospital of Philadelphia, said in a statement. "In our study, we built the computational algorithms to leverage clinical data. We then used these tools to find the genetic cause for a patient's epilepsy."

He and his colleagues used whole-exome sequencing data from 314 individuals with developmental and epileptic encephalopathies to uncover 11 genes with de novo variants in two or more individuals, including the AP2M1 gene.

At the same time, using phenotypic data on these individuals adhering to the standard Human Phenotype Ontology format, the researchers calculated the phenotypic similarity among their set of patients. They found that the two patients sharing the c.508C>T de novo variant in AP2M1 had significantly similar phenotypes.

The researchers further uncovered two additional individuals with this variant in a cohort of 2,310 people with epilepsy. All four of these patients had global developmental delay, three of the four had atonic seizures and generalized epileptiform discharges on EEG, and two of the four also had an autism spectrum disorder diagnosis.

The AP2M1 gene, the researchers noted, is highly expressed in the central nervous system and encodes an essential subunit of the AP-2 complex, which is involved in clathrin-mediated endocytosis. This process is a key part of synaptic vesicle recycling in mammals.

AP2M1, they added, appears intolerant of variation on the population level, as there are fewer-than-expected loss-of-function variants within it in the ExAC database. When the researchers modeled how the de novo variant they uncovered would affect the AP-2 complex, they found that the variant led to increased entropy in both the open and closed protein complex configurations, as compared to wild type. They suggested that this instability could be due to the variant introducing a hydrophobic tryptophan in the place of a charged arginine.

Additionally, both HeLa cells depleted of the affected AP-2 subunit and mouse cells with the affected AP-2 subunit knocked out exhibited reduced clathrin-mediated endocytosis. This indicated to the researchers that the variant might affect how AP-2 recognizes its cargo and, in turn, clathrin-mediated endocytosis.

The researchers further said this early disruption of in clathrin-mediated endocytosis could be a mechanism through which developmental and epileptic encephalopathies arise.

"This study emphasizes how the large amount of clinical information that is collected [on] our patients can facilitate gene discovery and enhance our understanding of a gene's function," CHOP's Ethan Goldberg added in a statement. "Increasingly, knowing how defects in a gene cause seizures allows us to better determine which medications might work and to develop new strategies to treat children with epilepsy."