Allelic strengths of encephalopathy-associatedUBA5variants correlate betweenin vivoandin vitroassays

Protein UFMylation downstream of the E1 enzyme UBA5 plays essential roles in development and ER stress. Variants in theUBA5gene are associated with developmental and epileptic encephalopathy 44 (DEE44), an autosomal recessive disorder characterized by early-onset encephalopathy, movement abnormalities, global developmental delay, intellectual disability, and seizures. DEE44 is caused by at least twelve different missense variants described as loss of function (LoF), but the relationships between genotypes and molecular or clinical phenotypes remains to be established. We developed a humanizedUBA5fly model and biochemical activity assays in order to describein vivoandin vitrogenotype-phenotype relationships across theUBA5allelic series.In vivo, we observed a broad spectrum of phenotypes in viability, developmental timing, lifespan, locomotor activity, and bang sensitivity. A range of functional effects was also observedin vitroacross comprehensive biochemical assays for protein stability, ATP binding, UFM1 activation, and UFM1 transthiolation. Importantly, there is a strong correlation betweenin vivoandin vitrophenotypes, establishing a classification of LoF variants into mild, intermediate, and severe allelic strengths. By systemically evaluatingUBA5variants acrossin vivoandin vitroplatforms, this study provides a foundation for more basic and translational UBA5 research, as well as a basis for evaluating current and future individuals afflicted with this rare disease.