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

Peroxisomal biogenesis disorders (PBD) are autosomal recessive disorders caused by loss-of-function mutations of one of the 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 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 gene mutations and utilized as a model organism. We generated flies replacing the coding sequence of our gene of interest with a promoter trap sequence. These cassettes simultaneously knock-out of the gene and knock-in a driver, ideal for making "humanized" flies in which the human gene can replace the fly loss. We assessed and 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 and variants in . We observed nearly complete phenotypic rescue of and loss when human or , respectively, were expressed. We also provide evidence for an allele severity spectrum in and in which some missense alleles, such as , are equally severe as early truncations, such as . We also observed that alleles associated with mild PBD, such as , show variability depending on the assay but do not fully rescue. Finally, alleles associated with atypical ataxia phenotypes, such as , can perform as well as , depending on the assay. Altogether, these lines effectively model the range of severity of peroxisomal biogenesis disorders.