Background: PTCHD1 is a susceptibility gene for autism spectrum disorder and intellectual disability. Its function in brain development and neurotransmission remains elusive. Studies have sought to characterize PTCHD1 function by elucidating its neural network of interacting proteins. However, given the current paucity of functional information, many PTCHD1 missense variants in clinical databases are classified as variants of uncertain significance (VUSs), severely limiting the health care resources available to patients and families.
Methods: A yeast 2-hybrid assay was used to identify synaptic PTCHD1-interacting proteins. Candidate binding partners were validated by cloning; transient overexpression in human embryonic kidney (HEK) 293T cells, followed by co-immunoprecipitation and immunoblotting; and immunocytochemistry in differentiated P19 cells. Site-directed mutagenesis was used to evaluate the pathogenicity of clinical missense variants, followed by transient overexpression and immunocytochemistry in non-neuronal (HEK293T) and neuronal (Neuro-2a cells) systems.
Results: A novel interaction was identified between the first lumenal loop of PTCHD1 and the SNARE-associated protein SNAPIN, which is implicated in synaptic vesicle exocytosis. Clinically associated missense variants within this region did not disrupt SNAPIN binding, indicating that the pathoetiology of these variants is unrelated to this interaction. However, 6 of the 12 missense variants tested exhibited pronounced retention within the endoplasmic reticulum and impaired neuronal and non-neuronal trafficking to the plasma membrane.
Conclusions: These data yield insights into the role of PTCHD1 in neurodevelopment and neurotransmission and suggest a neuropathological mechanism for missense variants. These findings provide a platform for diagnostic assay and VUS interpretation, allowing for clinical reclassification of these variants.
Keywords: Autism spectrum disorder; Clinical missense variant; Intellectual disability; Protein-protein interaction; SNARE complex; Transmembrane protein trafficking.
Loss-of-function variants in the X-linked PTCHD1 gene result in autism spectrum disorder and/or intellectual disability in males. However, missense variants in PTCHD1 are typically reported as variants of uncertain significance by diagnostic laboratories, in part due to the limited understanding of PTCHD1 protein function and the lack of a functional assay. Pastore et al. (article#) identify the SNARE-associated protein SNAPIN as a candidate protein interacting partner for PTCHD1 through PTCHD1 external loop 1. However, clinically reported missense variants in loop 1 do not impact the protein-protein interaction. Of 12 PTCHD1 missense variants tested, 6 exhibited pronounced retention within the endoplasmic reticulum and impaired neuronal and non-neuronal trafficking to the plasma membrane, suggesting a putative (and assayable) neuropathological mechanism.
© 2025 The Authors.