Pastore Stephen F, Xie Connie T Y, Derwish Roya, Muhammad Tahir, Blahova Tereza, El-Masri Sierra C, Frankland Paul W, Hamel Paul A, Vincent John B
Molecular Neuropsychiatry & Development Lab, Molecular Brain Science Research Department, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Toronto, Ontario, Canada.
Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada.
Biol Psychiatry Glob Open Sci. 2025 Mar 22;5(4):100492. doi: 10.1016/j.bpsgos.2025.100492. eCollection 2025 Jul.
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.
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.
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.
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.
是自闭症谱系障碍和智力残疾的一个易感基因。其在大脑发育和神经传递中的功能仍不清楚。研究试图通过阐明其相互作用蛋白的神经网络来表征PTCHD1的功能。然而，鉴于目前功能信息的匮乏，临床数据库中的许多PTCHD1错义变体被归类为意义未明的变体（VUS），严重限制了可用于患者及其家庭的医疗资源。
采用酵母双杂交试验来鉴定与突触PTCHD1相互作用的蛋白。通过克隆验证候选结合伙伴；在人胚肾（HEK）293T细胞中瞬时过表达，随后进行共免疫沉淀和免疫印迹；以及在分化的P19细胞中进行免疫细胞化学。使用定点诱变来评估临床错义变体的致病性，随后在非神经元（HEK293T）和神经元（Neuro-2a细胞）系统中进行瞬时过表达和免疫细胞化学。
在PTCHD1的第一个腔内环与SNARE相关蛋白SNAPIN之间鉴定出一种新的相互作用，SNAPIN与突触小泡胞吐作用有关。该区域内临床相关的错义变体并未破坏与SNAPIN的结合，表明这些变体的发病机制与这种相互作用无关。然而，所测试的12个错义变体中有6个在内质网中表现出明显的滞留，并损害了向质膜的神经元和非神经元转运。
这些数据揭示了PTCHD1在神经发育和神经传递中的作用，并提示了错义变体的神经病理机制。这些发现为诊断检测和VUS解释提供了一个平台，允许对这些变体进行临床重新分类。
