Functional Analysis of DNM1L Missense Mutations utilizing Tet-Off based neurodevelopmental model

Abstract

Mitochondria fission requires Drp1, a GTP hydrolyzing enzyme encoded by the DNM1L allele. Pathogenic mutations in DNM1L are associated with a wide range of neurological symptoms, from mild optic atrophy to severe developmental delay with seizures. Despite the expanding spectrum of DNM1L-related diseases, the molecular mechanisms underlying the genotype-phenotype correlation among DNM1L variant is unclear. To evaluate the neurodevelopmental impact of each DNM1L variants, we designed a Tet-Off system in human embryonic stem cells to avoid the lethal effect of DNM1L ablation. We transduced the Tet-Off system that can control ectopic DNM1L wild-type expression by doxycycline, and induced endogenous DNM1L knockout by CRISPR-Cas9. Clones were selected by puromycin and confirmed by sequencing. Doxycycline dependency of the constructed stem cell line was tested in RNA and protein levels. The effects of each variant will be investigated regarding Drp1 functions, especially mitochondria fission, after differentiating into neural progenitor cells and mature neurons. By expanding the comprehension of DNM1L-related disorders, this study may provide insights into therapeutic development and thus improve the quality of patients’ life.