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Abstract

Adaptor protein complex 4 (AP-4)-associated hereditary spastic paraplegia (AP-4-HSP) is caused by biallelic loss-of-function variants in AP4B1, AP4M1, AP4E1 or AP4S1 which constitute the four subunits of this obligate complex. While the diagnosis of AP-4-HSP relies on molecular testing, the interpretation of novel missense variants remains challenging. Here we address this diagnostic gap by using patient-derived fibroblasts to establish a functional assay that measures the subcellular localization of ATG9A, a transmembrane protein that is sorted by AP-4. Using automated high-throughput microscopy, we determine the ratio of the ATG9A fluorescence in the trans-Golgi-network versus cytoplasm and ascertain that this metric meets standards for screening assays (Z’-robust > 0.3, SSMD > 3). The ‘ATG9A ratio’ is increased in fibroblasts of 17 well-characterized AP-4-HSP patients (mean: 1.54 ± 0.13 vs. 1.21 ± 0.05 (SD) in controls) and receiver-operating-characteristic analysis demonstrates robust diagnostic power (AUC: 0.85, 95%CI: 0.849-0.852). Using fibroblasts from two individuals with atypical clinical features and novel biallelic missense variants of unknown significance in AP4B1, we show that our assay can reliably detect AP-4 function. Our findings establish the ‘ATG9A ratio’ as a diagnostic marker of AP-4-HSP.