Exome Sequencing Implicates Impaired GABA Signaling and Neuronal Ion Transport in Trigeminal Neuralgia

Abstract

Trigeminal neuralgia (TN) is a common, debilitating neuropathic face pain syndrome often resistant to therapy. The familial clustering of TN cases suggests that genetic factors play a role in disease pathogenesis. However, no unbiased, large-scale genomic study of TN has been performed to date. Analysis of 290 whole exome-sequenced TN probands, including 20 multiplex kindreds and 70 parent-offspring trios, revealed enrichment of rare, damaging variants in GABA receptor-binding genes in cases. Mice engineered with a TN-associated de novo mutation (p.Cys188Trp) in the GABA_A receptor Cl^- channel γ-1 subunit (GABRG1) exhibited trigeminal mechanical allodynia and face pain behavior. Other TN probands harbored rare damaging variants in Na⁺ and Ca⁺ channels, including a significant variant burden in the α-1H subunit of the voltage-gated Ca²⁺ channel Ca_v3.2 (CACNA1H). These results provide exome-level insight into TN and implicate genetically encoded impairment of GABA signaling and neuronal ion transport in TN pathogenesis.

Keywords: Genomics; Neuroscience; Structural Biology.

Graphical abstract

Figure 1

Pedigrees for 20 TN Familial Cases 20 familial cases with ≥2 members available for whole-exome sequencing (WES) are shown with sample IDs. Black circle/squares: Subjects with TN diagnosis. See also Table S1.

Figure 2

Damaging De Novo and Inherited Mutations in GABRG1 and TRAK1 in TN Probands (A) De novo and inherited mutations in GABRG1 and TRAK1. Pedigrees with Sanger-validated mutant bases marked on the chromatograms. (B) Mapping of GABRG1 and TRAK1 mutations. GABRG1 p.Cys188Trp and p.Tyr178His impact conserved residues at the neurotransmitter-gated ligand-binding domain of GABRG1. TRAK1 p.Glu798Lys affects a conserved residue of the second kinesin-binding Milton domain, whereas TRAK1 p.Arg124Gln maps to a conserved residue of the HAP1_N domain. (C) Structural modeling of GABRG1 p.Cys188Trp and p.Tyr178His mutations. p.Cys188 disulfide bonds with Cys202. Mutation to Trp188 disrupts this conserved disulfide bond with calculated ΔΔG = 3.8 kcal/mol. In the midst of surrounding hydrophobic residues, the side chain hydroxyl of Tyr178 hydrogen bonds with the guanidinium side chain of Arg166, stabilizing interactions between their adjacent β-sheets. Disruption of this hydrogen bond by Arg mutation to His is predicted to destabilize ΔΔG by 1.7 kcal/mol. (D) WT and mutant mice were examined for nocifensive withdrawal behavior following stimulation of the trigeminal nerve region in response to mechanical stimulation using a single von Frey filament (#7; 0.6 g). The y axis values represent average responses to three stimulations (on different days; +1 = presence of a stimulus-associated grooming response; −1 = absence of grooming behavior). The Mann-Whitney test was applied to assess statistical difference between WT and mutant mice. A Kruskal-Wallis test evaluated significant differences among all sub-groups: male and female WT and mutants. p value ∗ <0.05; ∗∗ <0.01; ∗∗∗ <1 × 10⁻³; ∗∗∗∗ <1 × 10⁻⁴. (E and F) Measurement of nociceptive withdrawal threshold using the using the Simplified Up-Down method (SUDO) (Bonin et al., 2014). (E) Graph showing withdrawal threshold results using an adaptation of the method to test in the region innervated by the trigeminal nerve (see Methods; Taylor et al. Pain, 2012). (F) Nociceptive withdrawal threshold in response to mechanical stimulation of the hind paw using calibrated von Frey filaments. p value ∗∗ <0.01. See also Figures S1 and S2; Tables S1, S3, S4, and S6; and Videos S1, S2, S3, S4, S5, S6, S7, and S8.

Figure 3

Gene Burden Analysis for Heterozygous Damaging Mutations and Mutation Mapping in Ca²⁺ Channels Encoded by CACNA1H and CACNA1F (A) Quantile-quantile plot of observed versus expected p values for damaging (LoF and D-mis) variants with MAF ≤1 × 10⁻⁴. The genome-wide significant gene CACNA1H is circled in red. The genome significance cutoff is 2.6 × 10⁻⁶, 0.05/19,347. (B and C) Mutation mapping of (B) CACNA1H and (C) CACNA1F. CACNA1H graph was adapted from Rzhepetskyy et al. (Rzhepetskyy et al., 2016); CACNA1F graph was modified from (Haeseleer et al., 2016). See also Figure S3 and Table S5.