Allelic strengths of encephalopathy-associated variants correlate between and assays.

Protein UFMylation downstream of the E1 enzyme UBA5 plays essential roles in development and ER stress. Variants in the gene 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 humanized fly model and biochemical activity assays in order to describe and genotype-phenotype relationships across the allelic series. , we observed a broad spectrum of phenotypes in viability, developmental timing, lifespan, locomotor activity, and bang sensitivity. A range of functional effects was also observed across comprehensive biochemical assays for protein stability, ATP binding, UFM1 activation, and UFM1 transthiolation. Importantly, there is a strong correlation between and phenotypes, establishing a classification of LoF variants into mild, intermediate, and severe allelic strengths. By systemically evaluating variants across and platforms, 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.