Allelic Strengths of Encephalopathy-Associated UBA5 Variants Correlate Between In Vivo and In Vitro Assays

Authors

Xueyang Pan
Albert N Alvarez
Mengqi Ma
Shenzhao Lu
Michael W Crawford
Lauren C Briere
Oguz Kanca
Shinya Yamamoto
David A Sweetser
Jenny L Wilson
Ruth J Napier
Jonathan N Pruneda
Hugo J Bellen

Publication Date

12-11-2023

Journal

eLife

DOI

10.7554/eLife.89891

PMID

38079206

PMCID

PMC10712953

PubMedCentral® Posted Date

12-11-2023

Published Open-Access

yes

Keywords

Humans, Brain Diseases, Intellectual Disability, Movement Disorders, Mutation, Missense, Ubiquitin-Activating Enzymes, Drosophila, Drosophila Proteins, D. melanogaster, Human

Abstract

Protein UFMylation downstream of the E1 enzyme UBA5 plays essential roles in development and endoplasmic reticulum stress. Variants in the UBA5 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 12 different missense variants described as loss of function (LoF), but the relationships between genotypes and molecular or clinical phenotypes remain to be established. We developed a humanized UBA5 fly model and biochemical activity assays in order to describe in vivo and in vitro genotype–phenotype relationships across the UBA5 allelic 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 observed in vitro across comprehensive biochemical assays for protein stability, ATP binding, UFM1 activation, and UFM1 transthiolation. Importantly, there is a strong correlation between in vivo and in vitro phenotypes, establishing a classification of LoF variants into mild, intermediate, and severe allelic strengths. By systemically evaluating UBA5 variants across in vivo and in vitro 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.