Abstract
Background Patched domain-containing 1 (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 healthcare resources available to patients and families.
Methods A yeast two-hybrid assay was used to identify synaptic PTCHD1-interacting proteins. Candidate binding partners were validated by cloning; transient over-expression in HEK293T cells, followed by co-immunoprecipitation and immunoblotting; and immunocytochemistry in differentiated P19 cells. To evaluate the pathogenicity of clinical missense variants, site-directed mutagenesis was employed, followed by transient over-expression 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, six 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 regarding 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 re-classification of these variants.
Competing Interest Statement
CAMH and J.B.V. hold intellectual property rights and a license for diagnostic testing for PTCHD1 with Quest Diagnostics. All other authors report no conflict of interest.
Footnotes
Additional data using a neuronal cell model system has been added. Two additional co-authors have been added Figures have been added/updated Supplementary Materials have been added/updated