2. Academic Validation
  3. High-throughput imaging of ATG9A distribution as a diagnostic functional assay for adaptor protein complex 4-associated hereditary spastic paraplegia

High-throughput imaging of ATG9A distribution as a diagnostic functional assay for adaptor protein complex 4-associated hereditary spastic paraplegia

  • Brain Commun. 2021 Sep 25;3(4):fcab221. doi: 10.1093/braincomms/fcab221.
Darius Ebrahimi-Fakhari 1 2,  Julian E Alecu 1,  Barbara Brechmann 1 3,  Marvin Ziegler 1 4,  Kathrin Eberhardt 1,  Hellen Jumo 1,  Angelica D'Amore 1,  Parham Habibzadeh 5,  Mohammad Ali Faghihi 6,  Jan L De Bleecker 7,  Sandrine Vuillaumier-Barrot 8,  Stéphane Auvin 9,  Filippo M Santorelli 10,  Sonja Neuser 11,  Bernt Popp 11,  Edward Yang 12,  Lee Barrett 1 13,  Alexandra K Davies 14,  Afshin Saffari 1 3,  Jennifer Hirst 15,  Mustafa Sahin 1 13
Affiliations

Affiliations

  • 1 Department of Neurology, The F.M. Kirby Neurobiology Center, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
  • 2 The Manton Center for Orphan Disease Research, Boston Children's Hospital, Boston, MA 02115, USA.
  • 3 Division of Neuropediatrics and Inherited Metabolic Diseases, Center for Pediatrics and Adolescent Medicine, University Hospital Heidelberg, 69120 Heidelberg, Germany.
  • 4 Ruprecht-Karls University Heidelberg, Medical School, 69120 Heidelberg, Germany.
  • 5 Persian BayanGene Research and Training Center, Shiraz University of Medical Sciences, 71347 Shiraz, Iran.
  • 6 Department of Psychiatry and Behavioral Sciences, University of Miami, Miami, FL 33136, USA.
  • 7 Department of Neurology, Ghent University Hospital, 9000 Ghent, Belgium.
  • 8 Department of Biochemistry, AP-HP, Bichat Hospital, 75018 Paris, France.
  • 9 Pediatric Neurology Department, AP-HP, Robert Debré Hospital, 75019 Paris, France.
  • 10 Department of Molecular Medicine, IRCCS Fondazione Stella Maris, 56128 Pisa, Italy.
  • 11 Institute of Human Genetics, University of Leipzig Medical Center, 04103 Leipzig, Germany.
  • 12 Division of Neuroradiology, Department of Radiology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
  • 13 Rosamund Stone Zander Translational Neuroscience Center, Boston Children's Hospital, Boston, MA 02115, USA.
  • 14 Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
  • 15 Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK.
PMID: 34729478 DOI: 10.1093/braincomms/fcab221
Abstract

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.

Keywords

adaptor protein complex 4; biomarker; functional assay; hereditary spastic paraplegia; high-throughput imaging.

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