Distinguishing PEX gene variant severity for mild, severe, and atypical peroxisome biogenesis disorders in Drosophila

Peroxisomal biogenesis disorders (PBD) are autosomal recessive disorders caused by loss-of-function mutations of one of the PEX genes responsible for peroxisomal formation. Impaired peroxisome assembly causes severe multisystemic failure with patient phenotypes ranging from epilepsy, liver disease, feeding issues, biochemical abnormalities, and neurodegeneration. Variants in the same PEX gene can produce wide differences in severity, ranging from individuals with death in the first year of life to adults with milder complications. To study this strong genotype-phenotype correlation, we selected specific human PEX gene mutations and utilized Drosophila as a model organism. We generated flies replacing the coding sequence of our Pex gene of interest with a KozakGAL4 (KZ) promoter trap sequence. These cassettes simultaneously knock-out of the Pex gene and knock-in a GAL4 driver, ideal for making "humanized" flies in which the human PEX gene can replace the fly loss. We assessed Pex2KZ and Pex16KZ lines in lifespan, bang sensitivity, and climbing assays and confirmed that these are strong loss-of-function alleles. In parallel, we generated human reference and variant UAS-cDNA lines of PEX2 and PEX16 variants in Drosophila. We observed nearly complete phenotypic rescue of Drosophila Pex2 and Pex16 loss when human PEX2Refor PEX16Ref, respectively, were expressed. We also provide evidence for an allele severity spectrum in PEX2 and PEX16 in which some missense alleles, such as PEX2C247R, are equally severe as early truncations, such as PEX2R119*. We also observed that alleles associated with mild PBD, such as PEX2E55K, show variability depending on the assay but do not fully rescue. Finally, alleles associated with atypical ataxia phenotypes, such as PEX16F332Del, can perform as well as PEX16Ref, depending on the assay. Altogether, these Drosophila lines effectively model the range of severity of peroxisomal biogenesis disorders.