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. 2023 Nov;64(11):2968-2981.
doi: 10.1111/epi.17746. Epub 2023 Aug 30.

De novo CLPTM1 variants with reduced GABAA R current response in patients with epilepsy

Nana Liu  1   2   3 , Jinliang Li  4 , Kai Gao  1   2   3   5 , Riley E Perszyk  6 , Jing Zhang  6 , Jingmin Wang  1   2   3   7 , Ye Wu  1   2   3 , Andrew Jenkins  6   8 , Hongjie Yuan  6   9 , Stephen F Traynelis  6   9 , Yuwu Jiang  1   2   3   5   10
Affiliations

De novo CLPTM1 variants with reduced GABAA R current response in patients with epilepsy

Nana Liu et al. Epilepsia. 2023 Nov.

Abstract

Objective: To investigate the clinical features and potential pathogenesis mechanism of de novo CLPTM1 variants associated with epilepsy.

Methods: Identify de novo genetic variants associated with epilepsy by reanalyzing trio-based whole-exome sequencing data. We analyzed the clinical characteristics of patients with these variants and performed functional in vitro studies in cells expressing mutant complementary DNA for these variants using whole-cell voltage-clamp current recordings and outside-out patch-clamp recordings from transiently transfected human embryonic kidney (HEK) cells.

Results: Two de novo missense variants related to epilepsy were identified in the CLPTM1 gene. Functional studies indicated that CLPTM1-p.R454H and CLPTM1-p.R568Q variants reduced the γ-aminobutyric acid A receptor (GABAA R) current response amplitude recorded under voltage clamp compared to the wild-type receptors. These variants also reduced the charge transfer and altered the time course of desensitization and deactivation following rapid removal of GABA. The surface expression of the GABAA R γ2 subunit from the CLPTM1-p.R568Q group was significantly reduced compared to CLPTM1-WT.

Significance: This is the first report of functionally relevant variants within the CLPTM1 gene. Patch-clamp recordings showed that these de novo CLPTM1 variants reduce GABAA R currents and charge transfer, which should promote excitation and hypersynchronous activity. This study may provide insights into the molecular mechanisms of the CLPTM1 variants underlying the patients' phenotypes, as well as for exploring potential therapeutic targets for epilepsy.

Keywords: channelopathy; epilepsy; loss-of-function; translational study; trio-based whole-exome sequencing.

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Conflict of interest statement

Conflict of interest

SFT is a member of the SAB for Eumentis Therapeutics and Sage Therapeutics, is a member of the Medical Advisory Board for the GRIN2B Foundation and the CureGRIN Foundation, is an advisor to GRIN Therapeutics, Combined Brain, and Neurocrine, is co-founder of NeurOp Inc. and AgriThera Inc., and is a member of the Board of Directors of NeurOp Inc. HY is PI on a research grant from Sage Therapeutics to Emory University School of Medicine. SFT and HY are co-inventors on Emory University-owned Intellectual Property that includes allosteric modulators of NMDA receptor function. All the authors confirmed that they have read the journal’s position on issues involved in ethical publications and that their studies were performed in accordance with these guidelines.

Figures

Figure 1.
Figure 1.. vEEG and brain MRI characteristics of the two unrelated patients with CLPTM1 variants.
(A) a1: the vEEG of Patient-1 at 9 months old with CLPTM1, c.1361G>A: p.Arg>454His (R454H) variant indicates plenty of 2.5–3.5-Hz slow waves in the bilateral frontal pole, the frontal and right central regions, and the frontal–temporal region during the sleep period, noticeably on the right at 9 months old. a2: the vEEG of Patient-1 at 10 months old. a3: the MRI of the Patient 1 at 9 months old showing slightly wider extra-cerebral space, longer T2 signal of the white matter located in the posterior cerebral ventricle, and myelination changes in the terminal region. (B) b1,2: the vEEG at 1 year and 3 months old (b1) and at 2 years old (b2) of the Patient-2 with CLPTM1, c.1703G>A: p.Arg>568Gln (R568Q) variant, indicated a high degree of disorder (top) and spasm and tonic spasm (bottom). b3: the MRI of the Patient-2 at 16 months showed wider and deeper bilateral cerebral hemisphere sulcus, wider cerebral ventricle, and white matter dysplasia.
Figure 2.
Figure 2.. De novo CLPTM1 variants and missense tolerance analysis of CLPTM1.
(A) Sanger sequencing results of Patient-1 with CLPTM1, c.1361G>A: p.Arg>454His (p.R454H) variant. (B) Sanger sequencing results of Patient-2 with CLPTM1, c.1703G>A: p.Arg>568Gln (p.R568Q) variant. (C) Residues R454H and R568Q were highly conserved across non-human species. *, identical; □, highly conservative; △, conservative. (D) The 1DMTR of CLPTM1 (sequence NM_001294.4), using a 21 residue smoothing window, highlighting the variants R454H and R568Q. (E) The 3DMTR of CLPTM1, using the closest 21 residues, highlighting the variants R454H and R568Q. The AlphaFold putative structure prediction was used to determine the 3DMTR for CLPTM1 (see Supplemental Figure S1 for structural details).
Figure 3.
Figure 3.. Effects of the two CLPTM1 variants on GABAAR current response under whole-cell voltage clamp current recording.
(A) Representative GABA-evoked current traces of CLPTM1-WT (black), CLPTM1-p.R454H (red), and CLPTM1-p.R568Q (blue) were recorded by applying 1 mM of GABA for 5 seconds at holding potential −60 mV. (B) Summary of the effects of the CLPTM1 variants on GABA-evoked peak currents. (C) Summary of the effects of the CLPTM1 variants on GABA-evoked peak current density. (D) Summary of the degree of desensitization (ratio of steady-state currents to peak currents). *p < 0.05; **p < 0.01, one-way ANOVA with Dunnett’s multiple comparison test.
Figure 4.
Figure 4.. Effects of the two CLPTM1 variants on GABAAR current charge transfer under outside-out patch-clamp recording.
(A) Representative GABA-evoked current traces of CLPTM1-WT (black), CLPTM1-p.R454H (red), and CLPTM1-p.R568Q (blue) were recorded by applying 1 mM of GABA at holding potential −60 mV. (B) Representative GABA-evoked current traces of the three groups at holding potential +40 mV. (C, E) Summary of the charge transfer from the GABA-evoked peak currents at −60 mV (C) and +40 mV (E). (D, F) Summary of the charge transfer from the GABA-evoked steady-state currents at −60 mV (D) and +40 mV (F). *p < 0.05; **p < 0.01, one-way ANOVA with Dunnett’s multiple comparison test.
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