| Title: |
High-throughput imaging of ATG9A distribution as a diagnostic functional assay for adaptor protein complex 4-associated hereditary spastic paraplegia. |
| Authors: |
Ebrahimi-Fakhari, Darius; Alecu, Julian E; Brechmann, Barbara; Ziegler, Marvin; Eberhardt, Kathrin; Jumo, Hellen; D'Amore, Angelica; Habibzadeh, Parham; Faghihi, Mohammad Ali; De Bleecker, Jan L; Vuillaumier-Barrot, Sandrine; Auvin, Stéphane; Santorelli, Filippo M; Neuser, Sonja; Popp, Bernt; Yang, Edward; Barrett, Lee; Davies, Alexandra K; Saffari, Afshin; Hirst, Jennifer; Sahin, Mustafa |
| Source: |
essn: 2632-1297 ; nlmid: 101755125 |
| Publication Year: |
2022 |
| Collection: |
Apollo - University of Cambridge Repository |
| Subject Terms: |
hereditary spastic paraplegia; adaptor protein complex 4; functional assay; high-throughput imaging; biomarker |
| Description: |
Adaptor protein complex 4-associated hereditary spastic paraplegia 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 adaptor protein complex 4-associated hereditary spastic paraplegia 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 adaptor protein complex 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'-factor robust >0.3, strictly standardized mean difference >3). The 'ATG9A ratio' is increased in fibroblasts of 18 well-characterized adaptor protein complex 4-associated hereditary spastic paraplegia patients [mean: 1.54 ± 0.13 versus 1.21 ± 0.05 (standard deviation) in controls] and receiver-operating characteristic analysis demonstrates robust diagnostic power (area under the curve: 0.85, 95% confidence interval: 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 adaptor protein complex 4 function. Our findings establish the 'ATG9A ratio' as a diagnostic marker of adaptor protein complex 4-associated hereditary spastic paraplegia. |
| Document Type: |
article in journal/newspaper |
| File Description: |
application/pdf |
| Language: |
English |
| Relation: |
PMC8557665; https://www.repository.cam.ac.uk/handle/1810/332224 |
| DOI: |
10.17863/CAM.79670 |
| Availability: |
https://www.repository.cam.ac.uk/handle/1810/332224; https://doi.org/10.17863/CAM.79670 |
| Rights: |
Attribution-NonCommercial 4.0 International ; https://creativecommons.org/licenses/by-nc/4.0/ |
| Accession Number: |
edsbas.4F79C7 |
| Database: |
BASE |