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. 2013 Sep 5;93(3):496-505.
doi: 10.1016/j.ajhg.2013.07.014. Epub 2013 Aug 29.

De Novo mutations in GNAO1, encoding a Gαo subunit of heterotrimeric G proteins, cause epileptic encephalopathy

Kazuyuki Nakamura  1 , Hirofumi Kodera, Tenpei Akita, Masaaki Shiina, Mitsuhiro Kato, Hideki Hoshino, Hiroshi Terashima, Hitoshi Osaka, Shinichi Nakamura, Jun Tohyama, Tatsuro Kumada, Tomonori Furukawa, Satomi Iwata, Takashi Shiihara, Masaya Kubota, Satoko Miyatake, Eriko Koshimizu, Kiyomi Nishiyama, Mitsuko Nakashima, Yoshinori Tsurusaki, Noriko Miyake, Kiyoshi Hayasaka, Kazuhiro Ogata, Atsuo Fukuda, Naomichi Matsumoto, Hirotomo Saitsu
Affiliations

De Novo mutations in GNAO1, encoding a Gαo subunit of heterotrimeric G proteins, cause epileptic encephalopathy

Kazuyuki Nakamura et al. Am J Hum Genet. .

Abstract

Heterotrimeric G proteins, composed of α, β, and γ subunits, can transduce a variety of signals from seven-transmembrane-type receptors to intracellular effectors. By whole-exome sequencing and subsequent mutation screening, we identified de novo heterozygous mutations in GNAO1, which encodes a Gαo subunit of heterotrimeric G proteins, in four individuals with epileptic encephalopathy. Two of the affected individuals also showed involuntary movements. Somatic mosaicism (approximately 35% to 50% of cells, distributed across multiple cell types, harbored the mutation) was shown in one individual. By mapping the mutation onto three-dimensional models of the Gα subunit in three different complexed states, we found that the three mutants (c.521A>G [p.Asp174Gly], c.836T>A [p.Ile279Asn], and c.572_592del [p.Thr191_Phe197del]) are predicted to destabilize the Gα subunit fold. A fourth mutant (c.607G>A), in which the Gly203 residue located within the highly conserved switch II region is substituted to Arg, is predicted to impair GTP binding and/or activation of downstream effectors, although the p.Gly203Arg substitution might not interfere with Gα binding to G-protein-coupled receptors. Transient-expression experiments suggested that localization to the plasma membrane was variably impaired in the three putatively destabilized mutants. Electrophysiological analysis showed that Gαo-mediated inhibition of calcium currents by norepinephrine tended to be lower in three of the four Gαo mutants. These data suggest that aberrant Gαo signaling can cause multiple neurodevelopmental phenotypes, including epileptic encephalopathy and involuntary movements.

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Figures

Figure 1
Figure 1
De Novo GNAO1 Mutations in Individuals with Epileptic Encephalopathy Schematic representation of GNAO1, including two transcript variants: transcript variant 1 (RefSeq NM_020988.2) with nine exons and transcript variant 2 (RefSeq NM_138736.2) with eight exons. The UTRs and coding regions are shown in white and black rectangles, respectively. Three mutations occurred in common exons of two transcript variants, and one mutation occurred uniquely in transcript variant 1. Note that the electropherogram of individual 2 suggested mosaicism of the c.521A>G mutation, and a heterozygous C>T change (rs1065375) was clearly demonstrated. All mutations caused substitution or deletion of evolutionarily conserved amino acids. Homologous sequences were aligned with the use of the CLUSTALW web site.
Figure 2
Figure 2
EEG and Brain MRI Features of Individuals with GNAO1 Mutations (A and B) Interictal EEG of individual 3. A suppression-burst pattern was observed at 2 months of age (A), and transition to hypsarrhythmia was observed at 4 months (B). (C) Interictal EEG of individual 2 shows a suppression-burst pattern at 2 months. (D) Interictal EEG of individual 4 shows a diffuse spike- or sharp-and-slow-wave complex at 5 years. (E–I) T2-weighted axial images through the basal ganglia (E, H, and I) and T1-weighted axial (F) and sagittal (G) images. Individual 1 showed cerebral atrophy at 5 years and 6 months (E). Individual 2 showed delayed myelination and thin corpus callosum at 10 months (F and G). Individual 3 showed normal appearance at 3 months (H). Individual 4 showed reduced white matter at 7 years (I).
Figure 3
Figure 3
Localization of V5-Tagged Gαo1 Proteins in N2A Cells Localization of WT and five altered Gαo1 proteins in N2A cells. The WT and p.Gly203Arg and p.Gly203Thr altered proteins were localized to the cell periphery. In contrast, the p.Thr191_Phe197del protein was localized to the cytosolic compartment. The other p.Asp174Gly and p.Ile279Asn proteins were localized to the cell periphery but were also observed in the cytosol. The scale bars represent 10 μm.
Figure 4
Figure 4
Structural Consideration of the Gα Amino Acid Substitutions in Some Complexed States (A) Map of the amino acid substitution sites on the crystal structures of Gα-containing complexes: the GDP-bound inactive Gαiβγ heterotrimer (left), the nucleotide-free Gαsβγ in complex with an agonist-occupied monomeric β2AR (center), and the GDP+AlF4−-bound Gαq in complex with its effector PLCβ (right). Molecular structures are shown as space-filling representations (from PyMOL). Gα, Gβ, and Gγ subunits are colored green, yellow, and pink, respectively, and the switch I and switch II regions in the Gα subunit are in light green. The β2AR (center) and PLCβ (right) molecules are colored brown. The substituted sites are shown in red, and the indicated amino acid numbers correspond to human Gαo1 and, in parentheses, rat Gαi1 (UniProtKB/Swiss-Prot P10824) (left), bovine Gαs (UniProtKB/Swiss-Prot P04896) (center), and mouse Gαq (UniProtKB/Swiss-Prot P21279) (right). The illustrations above each model show the orientation of each subunit and the bound molecules. (B) The free-energy change after each of the amino acid substitutions was estimated from calculations using FoldX software. Each error bar represents an average value with a SD.
Figure 5
Figure 5
Evaluation of Gαo-Mediated Signaling in NG108-15 Cell Calcium-Current Generation (A) Representative traces of voltage-gated calcium currents generated in NG108-15 cells expressing WT (left) or p.Thr191_Phe197del altered (right) Gαo1. Black and red traces represent the currents before and 3 min after application of 10 μM norepinephrine, respectively. (B) Current densities of the calcium currents before norepinephrine treatment in cells expressing WT or altered Gαo1. Scatter plots represent the densities in individual cells. Red squares and bars represent the means and SEMs, respectively, of the densities in individual cell groups (WT, n = 8; p.Gly203Thr, n = 7; p.Asp174Gly, n = 8; p.Thr191_Phe197del, n = 7; p.Ile279Asn, n = 7). Compared with that in cells expressing WT Gαo1, the current density in cells expressing p.Thr191_Phe197del increased significantly (∗p < 0.05 by Dunnett’s post hoc test). The densities in the cells expressing other altered proteins did not vary significantly. (C) Comparison of norepinephrine-induced inhibition of calcium currents in cells expressing altered Gαo1. Each error bar represents the mean and SEM of the percent decrease in current density induced by application of 10 μM norepinephrine. Paired t tests indicated that the inhibition induced by norepinephrine was significant in cells expressing WT (n = 8) and p.Gly203Thr (n = 7), p.Asp174Gly (n = 8), and p.Ile279Asn (n = 7) altered proteins (∗∗p < 0.01 and ∗p < 0.05), but not in cells expressing p.Thr191_Phe197del (n = 7). Although there was some tendency for decreased inhibition in cells expressing altered proteins, the tendency did not reach statistical significance compared with that in WT-expressing cells (p = 0.41 by ANOVA).
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