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

Judith Halewa,Martine Raynaud,Marianne Till,Bénédicte Gérard,Manon Dixneuf,Valérie Chune,Annick Toutain,Nicolas Chatron,Jamal Ghoumid,Frédéric Laumonnier,Lyse Ruaud,Sylviane Marouillat,Sarah Grotto,Dévina C Ung,Thomas Smol,Rose-Anne Thépault ,Marie‐Laure Vuillaume ,Nathalie Costedoat‐Chalumeau ,Alain Verloès ,Gaëtan Lesca

doi:10.1002/humu.24208

Abstract

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.

Highlights

From the last few decades, the molecular study of neurodevelopmental disorders revealed the involvement of numerous genes essential in neuronal developmental processes like morphogenesis or neuronal plasticity and synaptogenesis (Bourgeron, 2015; Gilman et al, 2011; Krishnan et al, 2016; Laumonnier et al, 2007; Moyses‐Oliveira et al, 2020; Parenti et al, 2020)
Fixed cells were blocked and permeabilized by a DPBS solution with 10% of Donkey serum (DS; Sigma‐Aldrich) and 0.2% Triton X‐100 (Sigma‐Aldrich) for 1 h at room temperature (RT)
The p.(Leu73Phe), p.(Ile173Val), p.(Val195Ile), p.(His359Arg), p.(Ala470Asp), and p.(Glu479Gly) mutations were described in patients with autism spectrum disorder (ASD), intellectual disability (ID), or other neurodevelopmental disorder (NDD) (Noor et al, 2010), the p.(Ser51Asn) variant was reported in a patient with ID (Torrico et al, 2015), and the p.(Lys181Thr) variant was identified by whole‐exome sequencing in a patient with frontal lissencephalic cortical dysplasia and seizures (Karaca et al, 2015; Table 1)

Summary

| BACKGROUND

From the last few decades, the molecular study of neurodevelopmental disorders revealed the involvement of numerous genes essential in neuronal developmental processes like morphogenesis or neuronal plasticity and synaptogenesis (Bourgeron, 2015; Gilman et al, 2011; Krishnan et al, 2016; Laumonnier et al, 2007; Moyses‐Oliveira et al, 2020; Parenti et al, 2020). Microdeletions and mutations in the X‐chromosomal PTCHD1 (patched domain containing 1) gene were described in patients with autism spectrum disorders (ASDs) and/or intellectual disability (ID; Chaudhry et al, 2015; Filges et al, 2011; Marshall et al, 2008; Noor et al, 2010). Single nucleotide variants (SNVs), including missense, nonsense or truncating mutation in PTCHD1 have been highlighted in patients with NDD. Little is known about the pathogenic evaluation of PTCHD1 missense variants using biological in vitro experiments, which leads to significant issues in genetic medical diagnosis. Besides the increasing number of pathogenic microdeletions and truncating mutations of PTCHD1 causing ASD and ID, our data provide further evidence of the role of PTCHD1 in neurodevelopmental disorders, with the growing contribution of missense mutations leading to loss‐of‐function consequences

| METHODS

| RESULTS

| DISCUSSION