Molecular and Regulatory Characterization of the Autism and Intellectual Disability Susceptibility Gene PTCHD1

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social deficiencies, circumscribed patterns of behaviour, stereotypy, and hyper- or hypo-reactivity to sensory stimuli. Nearly half of individuals with ASD exhibit comorbidity with intellectual disability (ID), which is typified by a low intelligence quotient and impairments in adaptive behaviours. Genetics are strongly implicated in the pathoetiologies of both ASD and ID. On Xp22.11, Patched domain-containing 1 (PTCHD1) (OMIM: 300828) has been identified as a highly-penetrant susceptibility gene for ASD and ID, with deleterious alleles consistently transmitted from unaffected maternal carriers to affected males. PTCHD1 contains three exons and two predicted protein-coding transcripts: 1) PTCHD1-a (exons 1-3), expressed in the brain and numerous peripheral tissues; and 2) shorter brain-specific PTCHD1-c (exons 1 and 3). The most prevalent mouse model to study the neurobiological consequences of Ptchd1 loss-of-function involves excision of exon 2 (Ptchd1Δ2), restricting expression to Ptchd1-c. This transcript contains a premature truncating codon in exon 3 (p.Leu118Valfs*51) predicted to disrupt the final 771 amino acids of Ptchd1. Consequently, Ptchd1Δ2 mice exhibit cognitive impairment, motor deficits, and neurobehavioural changes; although notably, they display neither social deficits nor stereotypy. Remarkably, an alternative mouse model developed collaboratively at CAMH and The Hospital for Sick Children in Toronto, containing an indel that disrupts a long open reading frame in exon 3 (Ptchd1Δ3) possesses the same behaviours as Ptchd1Δ2 mice, in addition to presenting with both social deficits and stereotypy. To investigate this, the first chapter of this dissertation attempts to delineate a molecular mechanism to account for this phenotypic disparity between Ptchd1Δ2 and Ptchd1Δ3 mice. Studies of embryonic and early postnatal mice have revealed that expression of Ptchd1 exhibits both brain sub-region and temporal fluctuations during neurodevelopment. Furthermore, several non-coding sequence variants at the PTCHD1 locus have been associated with ASD, suggesting that PTCHD1 may be a dosage-sensitive gene necessary for proper neurodevelopment and neurotransmission. Therefore, the second chapter of this dissertation characterizes regulatory segments and motifs within the Ptchd1 upstream region, as well as identifies and validates a novel downstream regulatory region. In silico analyses predict PTCHD1 to be a polytopic protein that contains 12 transmembrane domains, which are organized into two sterol-sensing domain-like modules, two large lumenal loops, and a C-terminal PDZ-binding domain. Functionally, exogenously expressed PTCHD1 has been observed to localize to the post-synaptic density (PSD) in neurons, and to interact with PSD proteins and bind cholesterol in vitro. Despite these findings, a comprehensive neuronal function of PTCHD1 has yet to be elucidated. From a clinical diagnostic perspective, because of this paucity of functional information, a significant number of PTCHD1 missense variants have been identified in patients with neurodevelopmental disorders, and have been classified as variants of uncertain significance for PTCHD1. Consequently, the third chapter of this dissertation evaluates processing within the rough endoplasmic reticulum, plasma membrane trafficking, and protein-protein interactions of selected clinically reported PTCHD1 missense variants. Collectively, these data have clarified our understanding of presumptive protein production in Ptchd1 loss-of-function mouse models; PTCHD1 neuronal expression and regulation; and the stability, processing, and localization of PTCHD1 missense variants reported by diagnostic clinics.

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