The mechanisms that underlie the extensive phenotypic diversity in genetic disorders are poorly understood. Here, we develop a large-scale assay to characterize the functional valence (gain or loss-of-function) of missense variants identified in UBE3A, the gene whose loss-of-function causes the neurodevelopmental disorder Angelman syndrome. We identify numerous gain-of-function variants including a hyperactivating Q588E mutation that strikingly increases UBE3A activity above wild-type UBE3A levels. Mice carrying the Q588E mutation exhibit aberrant early-life motor and communication deficits, and individuals possessing hyperactivating UBE3A variants exhibit affected phenotypes that are distinguishable from Angelman syndrome. Additional structure-function analysis reveals that Q588 forms a regulatory site in UBE3A that is conserved among HECT domain ubiquitin ligases and perturbed in various neurodevelopmental disorders. Together, our study indicates that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggests that functional variant analysis can help delineate mechanistic subtypes in monogenic disorders.

UBE3A is a HECT (homologous to E6AP C‐terminus) domain E3 ubiquitin ligase that targets substrate proteins for degradation through the ubiquitin‐proteasome pathway. The UBE3Agene is of unique interest for its gene dosage‐dependent effect in the developing brain: Precise deletion or null mutation of the maternal copy of UBE3A causes a severe intellectual disability known as Angelman syndrome; meanwhile, duplication or triplication of the gene region in which UBE3A resides is linked to a prevalent syndromic form of autism known as Dup15q syndrome. However, little is known about the effects of missense variants which cause a single amino acid change in the enzyme, and prediction of disease outcomes for a given variant remains a challenge. Here, we pose that investigating variants’ effects on UBE3A functional activity levels is critical for predicting disease. In order to identify if precise mutations in UBE3A are sufficient to drive disease, we devised a high‐throughput assay to screen the functional consequence of UBE3A missense variants. We screened over 150 variants and identified distinct functional classes of UBE3A mutants based on their effect on enzymatic activity. Importantly, we identified over a dozen novel gain‐of‐functionvariants that aberrantly hyperactivate UBE3A enzyme activity. Through collaborations with clinical centers, we confirm that individuals possessing hyperactivating UBE3A variants exhibited phenotypes that were distinguishable from Angelman. Mice carrying a specific hyperactivating mutation on the maternal allele exhibited aberrant motor and early communication defects, as well as microcephaly. Finally, we mapped the results of our screen to the UBE3A protein structure to reveal a previously‐undefined allosteric regulatory exosite within the catalytic domain that we show to act as a charge‐dependent regulator of enzymatic activity. We found additional HECT domain enzymes to possess disease‐associated variants within their exosites, suggesting that exosite dysfunction is a common mechanism underlying a set of neurodevelopmental disorders. Together, our study indicates that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggests that deep structure‐functional analysis of protein variants can uncover disease‐relevant regulatory mechanisms.

UBE3A is a HECT (homologous to E6AP C‐terminus) domain E3 ubiquitin ligase that targets substrate proteins for degradation through the ubiquitin‐proteasome pathway. Deletion or null mutation of the maternal UBE3A gene causes Angelman syndrome, a severe intellectual disability disorder also characterized by epilepsy, motor deficits, and an unusually happy disposition. Hundreds of single amino acid variants of UBE3A have been reported in the literature, but the biological significance for most these variants are unknown. Here, we devised a high‐throughput assay to screen the functional consequence of UBE3A missense variants to identify precise mutations that drive disease. We screened over 150 UBE3A variants and identified distinct functional classes of UBE3A mutants, including over a dozen gain‐of‐function variants previously not known to exist. Mice carrying a specific hyperactivating mutation in UBE3A exhibited aberrant motor and communication deficits, and analysis of patient data showed that UBE3A hyperactivation causes a neurodevelopmental disorder that is distinct from Angelman syndrome. Finally, our detailed structure‐function analysis revealed a novel allosteric regulatory site in UBE3A that is perturbed by both gain and loss‐of‐function mutations. Altogether, our study demonstrates that functional variant analysis can delineate disease subtypes and provide deep structure‐functional data to uncover disease‐relevant regulatory mechanisms.