High-Throughput Imaging of ATG9A Distribution as a Diagnostic Functional Assay for Adaptor Protein Complex 4: Associated Hereditary Spastic Paraplegia (AP-4-HSP)

Background/Purpose: Adaptor protein complex 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.

Methods: A combination of clinical and molecular characterization including in silico analyses and high-throughput microscopy in patient-derived fibroblasts.

Results: 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.

Conclusion: Our findings establish the “ATG9A ratio” in patient-derived fibroblasts as a diagnostic marker of AP-4-HSP, which expands the set of tools available to assess the functional impact of novel variants in AP-4 subunit genes.

^*Corresponding author.

Publication History

Article published online:
28 October 2021