Identification of disease-linked hyperactivating mutations in UBE3A through large-scale functional variant analysis

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.


Supplementary Note 1
We obtained additional information for two other unrelated individuals who possessed a T787A mutation.
Individual 3 was an adolescent found to have intractable epilepsy. Individual 3 was diagnosed with Lennox-Gastaut syndrome without status epilepticus. Individual 4 was an adult diagnosed with intractable migraine accompanied by localization-related epilepsy with complex partial seizures. In both cases, the T787A mutation was identified through a complete epilepsy panel, and we could only obtain limited phenotypic information about these individuals. Importantly, the parental origin of the mutation was not established in either individual. We also identified another mutation at this site (T787M) that raised UBE3A activity 795.38% ± 101.8 above WT levels (Table 1), but we were not able to obtain phenotypic information for this individual. Individual 8 was a child who possessed a L781H mutation that increased UBE3A activity 615.29% ± 72.7 above WT levels (Supplementary Data 1). The proband had a low APGAR score at birth with good recovery, and no prenatal, pregnancy, or delivery issues. There was no family history of neurodevelopmental disease.
Individual 8 was diagnosed initially with developmental delay with hypotonia, feeding issues, and gastroesophageal reflex disease. The individual's functional age was determined to be 15-18 months at the time of examination, and the individual displayed minimal dysmorphism and normal pre and postnatal growth.
Epilepsy was observed during infancy and consisted of daily events of behavior arrest, staring, and lip smacking, with occasional eye deviation. Tremulousness in the upper extremities was evident in childhood. Individual 7 exhibited autistic phenotypes including mild sensory issues with light, echolalia, and head banging, but these phenotypes were not consistent with Angelman syndrome. Sequencing confirmed that the L781H mutation was on the maternally-inherited allele. Individual 8 exhibited unusual levels of fatigue and subsequent metabolic studies indicated some mitochondrial dysfunction. Additional sequencing also identified a pathogenic variant in

STXBP1.
We obtained limited information for Individual 9. The proband possessed an A521T mutation that increased UBE3A activity 464.25% ± 40.0 above WT levels (Supplementary Data 1). The patient exhibited seizures and developmental delay. Sequencing analysis confirmed the mutation was maternally inherited. The mother was unaffected, but had a family history of seizures, suggesting she may have been a carrier for the mutation.
Finally, we identified two additional cases in which the parental origin could not be established. Individual 10 was an adolescent who possessed a R516W mutation that raised activity 812.28% ± 43.8 above WT UBE3A levels. The proband presented with feeding difficulties and began to exhibit staring spells as a child. Individual 10 was diagnosed initially with fetal alcohol syndrome. As a child, the patient's staring spells became frequent and abnormal EEG patterns were evident. The proband failed six different drug treatments to control seizures, and complex seizures including atonic and tonic/clonic seizures were present in childhood. Consistent cognitive delays were also noted without evidence of regression. The proband was not tested for autism due to the extent of the individual's intellectual disability. MRI studies showed that Individual 10 possessed a small right choroidal tissue cyst and moderate skeletal deformations including mild tibial torsion. Individual 10 was adopted and the parental origin of the UBE3A mutation could not be established. Notably, our screen also identified an additional mutation at this site (R516Q) that increased activity 288.95% ± 11.1 above WT enzyme levels. Individual 11 was a child who possessed a G755S mutation that raised UBE3A activity 177.36% ± 12.5 above WT enzyme levels.
This individual exhibited seizures and developmental delay. Sequencing analysis determined the mother was negative for the mutation, but it was unknown if the mutation was paternally inherited or de novo. Collectively, these data demonstrate that hyperactivating mutations in UBE3A are sufficient to cause a distinct disorder that manifests during early developmental periods.  (a) Mating schemes used to generate heterozygous mice with a maternally-inherited Q588E mutation (mQ588E) and a paternally-inherited Q588E mutation (pQ588E).

Supplementary
(b) Western blot analysis showing UBE3A (black arrowhead) and actin (red arrowhead) protein levels in the cortex from WT, mQ588E, and pQ588E mice. Three mice per genotype were analyzed in each blot.
(c) Individual and mean ± SE for righting times for WT (black) and mQ588E (gray) animals in the righting reflex assay. WT, n = 29 animals; mQ588E, n = 14 animals.
(d and e) Grasping reflexes measured for the forepaw and hindpaw of WT (black) and mQ588E (gray) animals.
Values are shown as the percent of tested animals exhibiting the grasping reflex; n.s., not significant. WT, n = 29 animals; mQ588E, n = 16 animals.
(f) Individual and mean ± SE times for WT (black) and mQ588E (gray) mice in the negative geotaxis assay are shown. Each animal was tested three times and the average of the three trials was used for analysis. WT, n = 29 animals; mQ588E, n = 19 animals.