Novel missense mutations in PTCHD1 alter its plasma membrane subcellular localization and cause intellectual disability and autism spectrum disorder.

The X-linked PTCHD1 gene, encoding a synaptic membrane protein, has been involved in neurodevelopmental disorders with the description of deleterious genomic microdeletions or truncating coding mutations. Missense variants were also identified, however, without any functional evidence supporting their pathogenicity level. We investigated 13 missense variants of PTCHD1, including eight previously described (c.152G>A,p.(Ser51Asn); c.217C>T,p.(Leu73Phe); c.517A>G,p.(Ile173Val); c.542A>C,p.(Lys181Thr); c.583G>A,p.(Val195Ile); c.1076A>G,p.(His359Arg); c.1409C>A,p.(Ala470Asp); c.1436A>G,p.(Glu479Gly)), and five novel ones (c.95C>T,p.(Pro32Leu); c.95C>G,p.(Pro32Arg); c.638A>G,p.(Tyr213Cys); c.898G>C,p.(Gly300Arg); c.928G>C,p.(Ala310Pro)) identified in male patients with intellectual disability (ID) and/or autism spectrum disorder (ASD). Interestingly, several of these variants involve amino acids localized in structural domains such as transmembrane segments. To evaluate their potentially deleterious impact on PTCHD1 protein function, we performed in vitro overexpression experiments of the wild-type and mutated forms of PTCHD1-GFP in HEK 293T and in Neuro-2a cell lines as well as in mouse hippocampal primary neuronal cultures. We found that six variants impaired the expression level of the PTCHD1 protein, and were retained in the endoplasmic reticulum suggesting abnormal protein folding. Our functional analyses thus provided evidence of the pathogenic impact of missense variants in PTCHD1, which reinforces the involvement of the PTCHD1 gene in ID and in ASD.

Phenotypic spectrum associated with PTCHD1 deletions and truncating mutations includes intellectual disability and autism spectrum disorder.

Studies of genomic copy number variants (CNVs) have identified genes associated with autism spectrum disorder (ASD) and intellectual disability (ID) such as NRXN1, SHANK2, SHANK3 and PTCHD1. Deletions have been reported in PTCHD1 however there has been little information available regarding the clinical presentation of these individuals. Herein we present 23 individuals with PTCHD1 deletions or truncating mutations with detailed phenotypic descriptions. The results suggest that individuals with disruption of the PTCHD1 coding region may have subtle dysmorphic features including a long face, prominent forehead, puffy eyelids and a thin upper lip. They do not have a consistent pattern of associated congenital anomalies or growth abnormalities. They have mild to moderate global developmental delay, variable degrees of ID, and many have prominent behavioral issues. Over 40% of subjects have ASD or ASD-like behaviors. The only consistent neurological findings in our cohort are orofacial hypotonia and mild motor incoordination. Our findings suggest that hemizygous PTCHD1 loss of function causes an X-linked neurodevelopmental disorder with a strong propensity to autistic behaviors. Detailed neuropsychological studies are required to better define the cognitive and behavioral phenotype.

Nonsynonymous Mutations in Intellectual Disability and Autism Spectrum Disorder Gene PTCHD1 Disrupt N-Glycosylation and Reduce Protein Stability.

PTCHD1 has been implicated in Autism Spectrum Disorders (ASDs) and/or intellectual disability, where copy-number-variant losses or loss-of-function coding mutations segregate with disease in an X-linked recessive fashion. Missense variants of PTCHD1 have also been reported in patients. However, the significance of these mutations remains undetermined since the activities, subcellular localization, and regulation of the PTCHD1 protein are currently unknown. This paucity of data concerning PTCHD1 prevents the effective evaluation of sequence variants identified during diagnostic screening. Here, we characterize PTCHD1 protein binding partners, extending previously reported interactions with postsynaptic scaffolding protein, SAP102. Six rare missense variants of PTCHD1 were also identified from patients with neurodevelopmental disorders. After modelling these variants on a hypothetical three-dimensional structure of PTCHD1, based on the solved structure of NPC1, PTCHD1 variants harboring these mutations were assessed for protein stability, post-translational processing, and protein trafficking. We show here that the wild-type PTCHD1 post-translational modification includes complex N-glycosylation and that specific mutant proteins disrupt normal N-link glycosylation processing. However, regardless of their processing, these mutants still localized to PSD95-containing dendritic processes and remained competent for complexing SAP102.

CRISPR-Cas9-generated PTCHD1 2489T>G stem cells recapitulate patient phenotype when undergoing neural induction.

An estimated 3.5%-5.9% of the global population live with rare diseases, and approximately 80% of these diseases have a genetic cause. Rare genetic diseases are difficult to diagnose, with some affected individuals experiencing diagnostic delays of 5-30 years. Next-generation sequencing has improved clinical diagnostic rates to 33%-48%. In a majority of cases, novel variants potentially causing the disease are discovered. These variants require functional validation in specialist laboratories, resulting in a diagnostic delay. In the interim, the finding is classified as a genetic variant of uncertain significance (VUS) and the affected individual remains undiagnosed. A VUS (PTCHD1 c. 2489T>G) was identified in a child with autistic behavior, global developmental delay, and hypotonia. Loss of function mutations in PTCHD1 are associated with autism spectrum disorder and intellectual disability; however, the molecular function of PTCHD1 and its role in neurodevelopmental disease is unknown. Here, we apply CRISPR gene editing and induced pluripotent stem cell (iPSC) neural disease modeling to assess the variant. During differentiation from iPSCs to neural progenitors, we detect subtle but significant gene signatures in synaptic transmission and muscle contraction pathways. Our work supports the causal link between the genetic variant and the child's phenotype, providing evidence for the variant to be considered a pathogenic variant according to the American College of Medical Genetics and Genomics guidelines. In addition, our study provides molecular data on the role of PTCHD1 in the context of other neurodevelopmental disorders.

Corrigendum: PTCHD1 gene mutation/deletion: the cognitive-behavioral phenotyping of four case reports.

[This corrects the article DOI: 10.3389/fpsyt.2023.1327802.].

PTCHD1 gene mutation/deletion: the cognitive-behavioral phenotyping of four case reports.

Introduction: X-linked PTCHD1 gene has recently been pointed as one of the most interesting candidates for involvement in neurodevelopmental disorders (NDs), such as intellectual disability (ID) and autism spectrum disorder (ASD). PTCHD1 encodes the patched domain-containing protein 1 (PTCHD1), which is mainly expressed in the developing brain and adult brain tissues. To date, major studies have focused on the biological function of the PTCHD1 gene, while the mechanisms underlying neuronal alterations and the cognitive-behavioral phenotype associated with mutations still remain unclear. Methods: With the aim of incorporating information on the clinical profile of affected individuals and enhancing the characterization of the genotype-phenotype correlation, in this study, we analyze the clinical features of four individuals (two children and two adults) in which array-CGH detected a PTCHD1 deletion or in which panel for screening non-syndromal XLID (X-linked ID) detected a PTCHD1 gene variant. We define the neuropsychological and psychopathological profiles, providing quantitative data from standardized evaluations. The assessment consisted of clinical observations, structured interviews, and parent/self-reported questionnaires. Results: Our descriptive analysis align with previous findings on the involvement of the PTCHD1 gene in NDs. Specifically, our patients exhibited a clinical phenotype characterized by psychomotor developmental delay- ID of varying severity. Interestingly, while ID during early childhood was associated with autistic-like symptomatology, this interrelation was no longer observed in the adult subjects. Furthermore, our cohort did not display peculiar dysmorphic features, congenital abnormalities or comorbidity with epilepsy. Discussion: Our analysis shows that the psychopathological and behavioral comorbidities along with cognitive impairment interfere with development, therefore contributing to the severity of disability associated with PTCHD1 gene mutation. Awareness of this profile by professionals and caregivers can promote prompt diagnosis as well as early cognitive and occupational enhancement interventions.

Generation of ventralized human thalamic organoids with thalamic reticular nucleus.

Human brain organoids provide unique platforms for modeling several aspects of human brain development and pathology. However, current brain organoid systems mostly lack the resolution to recapitulate the development of finer brain structures with subregional identity, including functionally distinct nuclei in the thalamus. Here, we report a method for converting human embryonic stem cells (hESCs) into ventral thalamic organoids (vThOs) with transcriptionally diverse nuclei identities. Notably, single-cell RNA sequencing revealed previously unachieved thalamic patterning with a thalamic reticular nucleus (TRN) signature, a GABAergic nucleus located in the ventral thalamus. Using vThOs, we explored the functions of TRN-specific, disease-associated genes patched domain containing 1 (PTCHD1) and receptor tyrosine-protein kinase (ERBB4) during human thalamic development. Perturbations in PTCHD1 or ERBB4 impaired neuronal functions in vThOs, albeit not affecting the overall thalamic lineage development. Together, vThOs present an experimental model for understanding nuclei-specific development and pathology in the thalamus of the human brain.

Disruption of DDX53 coding sequence has limited impact on iPSC-derived human NGN2 neurons.

BACKGROUND: The X-linked PTCHD1 locus is strongly associated with autism spectrum disorder (ASD). Males who carry chromosome microdeletions of PTCHD1 antisense long non-coding RNA (PTCHD1-AS)/DEAD-box helicase 53 (DDX53) have ASD, or a sub-clinical form called Broader Autism Phenotype. If the deletion extends beyond PTCHD1-AS/DDX53 to the next gene, PTCHD1, which is protein-coding, the individuals typically have ASD and intellectual disability (ID). Three male siblings with a 90 kb deletion that affects only PTCHD1-AS (and not including DDX53) have ASD. We performed a functional analysis of DDX53 to examine its role in NGN2 neurons. METHODS: We used the clustered regularly interspaced short palindromic repeats (CRISPR) gene editing strategy to knock out DDX53 protein by inserting 3 termination codons (3TCs) into two different induced pluripotent stem cell (iPSC) lines. DDX53 CRISPR-edited iPSCs were differentiated into cortical excitatory neurons by Neurogenin 2 (NGN-2) directed differentiation. The functional differences of DDX53-3TC neurons compared to isogenic control neurons with molecular and electrophysiological approaches were assessed. RESULTS: Isogenic iPSC-derived control neurons exhibited low levels of DDX53 transcripts. Transcriptional analysis revealed the generation of excitatory cortical neurons and DDX53 protein was not detected in iPSC-derived control neurons by western blot. Control lines and DDX53-3TC neurons were active in the multi-electrode array, but no overt electrophysiological phenotype in either isogenic line was observed. CONCLUSION: DDX53-3TC mutation does not alter NGN2 neuronal function in these experiments, suggesting that synaptic deficits causing ASD are unlikely in this cell type.

PTCHD1: Identification and Neurodevelopmental Contributions of an Autism Spectrum Disorder and Intellectual Disability Susceptibility Gene.

Over the last one and a half decades, copy number variation and whole-genome sequencing studies have illuminated the considerable genetic heterogeneity that underlies the etiologies of autism spectrum disorder (ASD) and intellectual disability (ID). These investigations support the idea that ASD may result from complex interactions between susceptibility-related genetic variants (single nucleotide variants or copy number variants) and the environment. This review outlines the identification and neurobiological characterization of two such genes located in Xp22.11, Patched domain-containing 1 (PTCHD1), and its antisense lncRNA PTCHD1-AS. Animal models of Ptchd1 disruption have recapitulated a subset of clinical symptoms related to ASD as well as to ID. Furthermore, these Ptchd1 mouse knockout studies implicate the expression of Ptchd1 in both the thalamic and the hippocampal brain regions as being crucial for proper neurodevelopment and cognitive function. Altered kynurenine metabolic signalling has been postulated as a disease mechanism in one of these animal studies. Additionally, ASD patient-derived induced pluripotent stem cells (iPSCs) carrying a copy number loss impacting the antisense non-coding RNA PTCHD1-AS have been used to generate 2D neuronal cultures. While copy number loss of PTCHD1-AS does not affect the transcription of PTCHD1, the neurons exhibit diminished miniature excitatory postsynaptic current frequency, supporting its role in ASD etiology. A more thorough understanding of risk factor genes, such as PTCHD1 and PTCHD1-AS, will help to clarify the intricate genetic and biological mechanisms that underlie ASD and ID, providing a foundation for meaningful therapeutic interventions to enhance the quality of life of individuals who experience these conditions.