Comprehensive Variant Effect Map of Parkin-Mediated Mitophagy in Parkinson’s Disease

Abstract The development of Parkinson’s disease (PD) has a substantial genetic basis. Variants in the PRKN gene account for roughly half of autosomal-recessive PD cases, yet most variants remain classified as variants of uncertain significance. PRKN encodes the E3 ubiquitin-protein ligase, Parkin, that plays a key role in mitochondrial quality control by initiating mitophagy. In this work, we introduce a multiplexed mitophagy assay and measure the activity of more than 99% of all possible single–amino acid substitution and nonsense variants of Parkin. We also demonstrate that misfolded, rapidly degraded variants autonomously trigger Parkin-independent mitophagy. The obtained activity landscape closely reflects known structural and functional aspects of Parkin while revealing new insights into specific functional effects across the protein. This dataset near-perfectly distinguishes pathogenic from benign variants and surpasses both computational predictors and abundance-based metrics in pathogenicity assessment. Finally, the data pinpoint hypomorphic variants that could be amenable to rescue in future personalized therapeutic approaches for PD. Competing Interest Statement K.L.-L. holds stock options in, receives sponsored research from, and is a consultant for Peptone Ltd. The remaining authors have no relevant financial or non-financial interests to disclose. - Abbreviations - ACT - activation element - AO - antimycin and oligomycin - AM - AlphaMissense - ARPD - autosomal-recessive Parkinson’s disease - AUC - area under the curve - Baf - Bafilomycin - DMEM - Dulbecco’s modified Eagle medium - Dox - doxycycline - DUBTAC - deubiquitylating targeted chimeras - FACS - fluorescence-activated cell sorting - FBS - fetal bovine serum - IBR - in-between RING - IQR - interquartile range - IRES - internal ribosomal entry site - MAGIC - mitochondria as guardian in cytosol - MAVE - multiplexed assay of variant effects - MED - median - OMM - outer mitochondrial membrane - PAP - peptide abundance predictor - PD - Parkinson’s disease - REP - Repressor element - ROC - Receiver Operating Characteristic - SD - standard deviation - pUb - phospho-ubiquitin - pUBL - phospho-UBL - Ub - ubiquitin - UBL - ubiquitin-like - VAMP-seq - variant abundance by massively parallel sequencing - VUS - variant of unknown significance - WT - wild-type.