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. 2017 Aug 22;89(8):762-770.
doi: 10.1212/WNL.0000000000004262. Epub 2017 Jul 26.

Movement disorder in GNAO1 encephalopathy associated with gain-of-function mutations

Huijie Feng  1 , Benita Sjögren  1 , Behirda Karaj  1 , Vincent Shaw  1 , Aysegul Gezer  1 , Richard R Neubig  2
Affiliations

Movement disorder in GNAO1 encephalopathy associated with gain-of-function mutations

Huijie Feng et al. Neurology. .

Abstract

Objective: To define molecular mechanisms underlying the clinical spectrum of epilepsy and movement disorder in individuals with de novo mutations in the GNAO1 gene.

Methods: We identified all GNAO1 mutations reported in individuals with epilepsy (early infantile epileptiform encephalopathy 17) or movement disorders through April 2016; 15 de novo mutant alleles from 25 individuals were introduced into the Gαo subunit by site-directed mutagenesis in a mammalian expression plasmid. We assessed protein expression and function in vitro in HEK-293T cells by Western blot and determined functional Gαo-dependent cyclic adenosine monophosphate (cAMP) inhibition with a coexpressed α2A adrenergic receptor.

Results: Of the 15 clinical GNAO1 mutations studied, 9 show reduced expression and loss of function (LOF; <90% maximal inhibition). Six other mutations show variable levels of expression but exhibit normal or even gain-of-function (GOF) behavior, as demonstrated by significantly lower EC50 values for α2A adrenergic receptor-mediated inhibition of cAMP. The GNAO1 LOF mutations are associated with epileptic encephalopathy while GOF mutants (such as G42R, G203R, and E246K) or normally functioning mutants (R209) were found in patients with movement disorders with or without seizures.

Conclusions: Both LOF and GOF mutations in Gαo (encoded by GNAO1) are associated with neurologic pathophysiology. There appears to be a strong predictive correlation between the in vitro biochemical phenotype and the clinical pattern of epilepsy vs movement disorder.

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Figures

Figure 1
Figure 1. Location and protein expression levels of human GNAO1 mutations related to epileptic encephalopathy
(A) Location of 15 mutations (G40R, G42R, D174G, T191_F197del, L199P, G203R, R209C, R209G, R209H, A227V, Y231C, E246K, N270H, F275S, and I279N) mapped on the Gαo amino acid sequence. (B, C) Representative Western blots of Gαo protein expression from HEK293T cells transiently transfected with each Gαo mutant. (D, E) Quantification of relative protein levels of each Gαo mutant compared to wild-type Gαo. Graphs are the result of 3 independent experiments and data are presented as mean ± SEM. **p < 0.01, ****p < 0.0001 using 1-way analysis of variance with Bonferroni post hoc test for pairwise comparison.
Figure 2
Figure 2. Effect on α2A adrenergic receptor (α2A AR)–mediated cyclic adenosine monophosphate (cAMP) inhibition by GNAO1 mutants
(A–C) Dose–response curves of representative GNAO1 mutants. (A) Dose–response curves of loss-of-function (LOF) and partial loss-of-function (PLOF) mutants (G40R, L199P, N270H, F275S, A227V, Y231C) show changes in cAMP production in response to the adenylate cyclase activator forskolin and α2 AR agonist UK14,304, compared to the positive control (wild-type [WT]) and negative control (pcDNA). (B) Dose–response curves of functioning Gαo mutants show changes in cAMP production in response to the α2 AR agonist UK14,304. All dose–response curves are shown in comparison with WT and G184S. (C) G42R displays a biphasic dose–response curve with cAMP inhibition at low concentrations (gain of function [GOF]), followed by enhancement of cAMP levels at higher concentrations of UK14,304. (D) Quantification of EC50 of functioning Gαo mutants. G42R, G203R, and E246K exhibit significantly increased potency for α2A AR–mediated cAMP inhibition similar to the known GOF mutation G184S. *p < 0.05, **p < 0.01, ***p < 0.001 using paired t test between WT and each mutant separately (figure e-4). (E) Percentage of maximum inhibition (n = 5) was normalized to pcDNA (0%; resulting in activation of cAMP) and WT (100%). ****p < 0.0001 using 1-way analysis of variance with Bonferroni post hoc test for pairwise comparison. Note that the maximum inhibition of G42R was calculated at UK14,304 of 15.8 nM. NF = normal function; PTX = pertussis toxin.
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