These findings expand the clinical and mechanistic spectrum of GlyR α2 variants, identify a key determinant of picrotoxin binding, and highlight unresolved roles of intracellular protein-protein interaction motifs in GlyR α2 function.
Abstract
Rare genetic variants in the glycine receptor (GlyR) α2 subunit gene (
GLRA2
) are associated with autism spectrum disorder, developmental delay, and intellectual disability, often accompanied by microcephaly, language delay or epilepsy. We report detailed structure-function analyses of nine previously uncharacterised GlyR α2 missense variants, including a novel
de novo
change (p.S285P) linked to epileptic encephalopathy. Using molecular modelling/dynamics simulations, electrophysiology, and immunocytochemistry, we assessed effects of GlyR α2 variants on agonist potency, efficacy, channel gating, and cell-surface trafficking. Five missense variants caused a partial
loss-of-function
via reduced glycine potency (p.F20S, p.A261T, p.R418Q), reduced glycine efficacy (p.F20S), or faster channel deactivation (p.R323C, p.P369T). By contrast, p.R225C abolished cell-surface expression resulting in a complete
loss-of-function
. The p.A261T variant also significantly reduced picrotoxin binding, resolving ambiguity in GlyR-PTX interaction models. Additional variants showed an
alteration-of-function
(p.I232M) or a
gain-of-function
(p.S285P), combining reduced glycine efficacy with increased potency and spontaneous leak currents. Two variants within the intracellular M3-M4 domain (p.R323C and p.P369T) had enhanced channel deactivation consistent with a
loss-of-function
, while p.P373L showed no detectable functional deficit. These findings expand the clinical and mechanistic spectrum of GlyR α2 variants, identify a key determinant of picrotoxin binding, and highlight unresolved roles of intracellular protein-protein interaction motifs in GlyR α2 function.
Background Purinergic P2X receptors (P2RX) play key roles in neuroinflammatory processes through ATP‐gated ion channel signaling. Dysregulation of P2RX receptor activity has been implicated in maternal immune activation, mitochondrial dysfunction, oxidative stress, and synaptic abnormalities associated with autism spectrum disorder (ASD). Despite increasing mechanistic evidence, the contribution of P2RX gene variants to ASD remains poorly understood. Methods We retrospectively evaluated whole‐exome sequencing (WES) data from individuals diagnosed with ASD according to DSM‐5 criteria. Variants in the P2RX1–P2RX7 genes were identified. Clinical, genetic, and in silico data were reviewed, and variants were classified according to ACMG/AMP guidelines. Results Eleven individuals were identified with heterozygous P2RX variants, most frequently in P2RX7 (54.5%), followed by P2RX5 (27.3%) and P2RX4 (18.2%). Core clinical features across all genes included ASD, intellectual disability (ID), and epilepsy. P2RX4 variants included an in‐frame deletion (p.Ile322del) and a truncating variant (p.Tyr366LeufsTer7), both observed in individuals with ASD and ID. P2RX5 variants comprised rare missense substitutions with variable in silico pathogenicity. Several P2RX7 variants, most notably the canonical splice‐site variant c.614 + 1G > A fulfilling ACMG/AMP PVS1 and PM2 criteria, were predicted to alter receptor function. Overall, in silico analyses indicated moderate to high predicted functional impact; however, most variants lacked segregation and functional validation data. Conclusion Our findings provide preliminary, exploratory observations suggesting that rare P2RX variants may represent biologically plausible candidates relevant to ASD‐related pathways; however, no conclusions regarding disease association or causality can be drawn. These findings should be considered hypothesis‐generating and require validation through larger controlled genetic studies and functional investigations.
Gül Ünsel-Bolat, H. Bolat· Human Mutation· 0 citations
The findings support the pathogenicity of this variant and further expand the pathogenic variant spectrum of the PPP2CA gene, and the observed genotype–phenotype correlation provides valuable information for prognosis and genetic counseling.
Lei Xu, Yanfeng Shen, Guixiang Zhang· Frontiers in Psychiatry· 0 citations
Tetratricopeptide Repeat Domain 14 plays a crucial role in RNA metabolism during neurodevelopment, and that the p.His30Arg variant impairs its function, possibly leading to a neurodevelopmental disorder within the lissencephaly spectrum.
S. R. Ahmad, M. Zeyaullah, Mohammad Suhail Khan et al.· Human Genetics· 0 citations
A family with two adult carriers of the heterozygous KCNQ2 p.(Arg214Trp) variant is reported, identified through whole-exome sequencing, including long-term follow-up into late adulthood, challenging the concept of KCNQ2 p.(Arg214Trp) as a self-limited epilepsy-associated variant and indicating a broader phenotypic spectrum.
P. Christova, M. Ostrožovičová, J. Neupauerová et al.· BMC Neurology· 0 citations
Disease variants in GABR genes encoding γ-aminobutyric acid type A receptor (GABAAR) subunits are major causes of developmental and epileptic encephalopathies (DEEs). There is no effective treatment for these DEEs, although the GABAAR is a major target for antiseizure drugs. We previously identified the therapeutic effect of 4-phenylbutyrate (PBA) in Gabrg2+/Q390X knockin DEE mice and in this study tested the effect of the drug in GABRA1 variants that encode the α1 subunit of GABAAR. We used a multidisciplinary approach including in silico structural modeling, flow cytometry, patch-clamp recordings and biochemistry in conjunction with differential tagging of the wildtype (WT) and the mutant alleles to evaluate the effect of PBA on rescue of GABAAR subunit expression, surface trafficking, and function in vitro in a heterologous HEK293T cell model and in vivo in Gabra1+/A322D mice. We found that the α1 subunit expression at both the total level and the cell surface was reduced when the variant α1 protein was present, suggesting reduced functional receptor availability on the cell membrane and synapse. Patch-clamp recordings identified that α1 variants reduced GABA-evoked current amplitude. In silico prediction indicated reduced protein stability for GABRA1 variants by negative ∆∆G values. PBA increased both total and surface expression of WT α1 and α1 variants and improved expression of both WT and variant α1 alleles when these were co-expressed. Importantly, PBA also increased the GABAAR expression in the cortex and thalamus of the Gabra1+/A322D mice. This study indicates that PBA is a promising treatment option for DEEs associated with GABRA1 mutations. Our previous work has demonstrated that PBA improves proteostasis by enhancing expression of the WT allele, repairing the mutant allele, and reducing endoplasmic reticulum stress in other DEEs associated with GABRG2 and SLC6A1 mutations. Importantly, it can mitigate seizures and improve neurobehavioral phenotypes at behavioral levels. Based on this and our previous work on GABRG2 and SLC6A1 mutations, we propose that PBA holds promise as a common medicine for multiple genetic neurologic disorders that share the proteostasis pathology with a broad clinical application in DEEs.
Ziang Song, Kirill Zavalin, W. Shen et al.· Cells· 0 citations
In two patients with SCN2A epileptic encephalopathy, treatment with personalized allele-selective antisense oligonucleotides led to a decrease in seizure frequency with a positive safety profile, and a pathway from n = 1 to n of more patients with SCN2A-RD and other monogenic disorders is provided.
Olivia Kim-Mcmanus, L. Mignon, J. Douville et al.· Nature Medicine· 2 citations