Severity of GNAO1-Related Disorder Correlates with Changes in G-Protein Function

Abstract

Objective: GNAO1-related disorders (OMIM #615473 and #617493), caused by variants in the GNAO1 gene, are characterized by developmental delay or intellectual disability, hypotonia, movement disorders, and epilepsy. Neither a genotype-phenotype correlation nor a clear severity score have been established for this disorder. The objective of this prospective and retrospective observational study was to develop a severity score for GNAO1-related disorders, and to delineate the correlation between the underlying molecular mechanisms and clinical severity.

Methods: A total of 16 individuals with GNAO1-related disorders harboring 12 distinct missense variants, including four novel variants (p.K46R, p.T48I, p.R209P, and p.L235P), were examined with repeated clinical assessments, video-electroencephalogram monitoring, and brain magnetic resonance imaging. The molecular pathology of each variant was delineated using a molecular deconvoluting platform.

Results: The patients displayed a wide variability in the severity of their symptoms. This heterogeneity was well represented in the GNAO1-related disorders severity score, with a broad range of results. Patients with the same variant had comparable severity scores, indicating that differences in disease profiles are not due to interpatient variability, but rather, to unique disease mechanisms. Moreover, we found a significant correlation between clinical severity scores and molecular mechanisms.

Interpretation: The clinical score proposed here provides further insight into the correlation between pathophysiology and phenotypic severity in GNAO1-related disorders. We found that each variant has a unique profile of clinical phenotypes and pathological molecular mechanisms. These findings will contribute to better understanding GNAO1-related disorders. Additionally, the severity score will facilitate standardization of patients categorization and assessment of response to therapies in development. ANN NEUROL 2023;94:987-1004.

Figure 1.. All variants exhibit an inhibited agonist response.

A. Location of each variant on Gαo (PDB: 3C7K). Regions are indicated as follows: Green – P-loop; Goldenrod – Switch I; Cyan – Switch II; Magenta – Switch III. B. Schematic of GPCR Signaling Assay. Dopamine stimulation induces the release of venus-Gβγ from Gαo, allowing venus-Gβγ to associate with NLuc-GRK3 and increase the BRET signal. C. Representative traces of variant dopamine BRET responses. D. The effect of GNAO1 variants on dopamine induced BRET signal. 0% response was defined as the amount of BRET signal upon dopamine activation of the Gαo-free control. 100% response was defined as the amount of BRET signal upon dopamine activation of the WT Gαo control. E. Schematic of Trimer formation assay. In the absence of Gαo, venus-Gβγ and NLuc-GRK3 have a high level of basal BRET. The binding of Gαo to Gβγ interferes with BRET between venus-Gβγ and NLuc-GRK3. F. The effect of GNAO1 variants on basal BRET. The amount of basal BRET without Gαo expressed was defined as 0. The amount of trimer formation is determined by subtracting the basal BRET of each variant from the Gαo-free control. G. Expression levels of each variant. Western blot analysis of each variant blotted with α-Gαo antibody.

Figure 2.. Some GNAO1 variants exhibit dominant negative activity.

A. Schematic of the Dominant Negative Activity Assay. Variants of Gαo are expressed alongside WT Gαo. Variants with dominant negative activity are able to suppress WT Gαo dopamine induced activation. B. Representative traces of dopamine BRET responses of GNAO1 variants expressed alongside WT Gαo. C. The effect of GNAO1 variants on WT Gαo dopamine induced BRET signal. Any condition with a max amplitude below the condition expressing pcDNA3.1+ alongside WT Gαo indicates that the variant has dominant negative activity. D. Schematic of the Receptor Interaction Assay. Dopamine stimulation induces the recruitment of G protein heterotrimer to the receptor where the SmBiT tag on the receptor can interact with the LgBiT tag on Gβ. This reconstitutes NLuc and gives a luminescent readout. E. Representative luminescence traces of the interaction of G protein heterotrimer with D2R receptor. F. The effect of GNAO1 variants on dopamine-induced receptor interaction with G protein heterotrimer. 0% interaction is defined as the amount of luminescence detected when the Gαo-free control was stimulated with dopamine. 100% interaction is defined as the amount of luminescence detected when WT Gαo was stimulated with dopamine.

Figure 3.. Correlations between Scores for GNAO1-related disorders severity score and Experimental Measurement.

A-F. Comparisons between clinical and experimental measurements do include repeated measures for patients with the same variant.

Figure 4.. Each GNAO1 variant presents a unique functional profile and can be placed on a spectrum of Loss-of-function/Dominant Negative activity.

A. Meta-analysis of all GNAO1 variants, combining the results of all functional experiments. B. Each variant can be placed on a spectrum with loss-of-function and dominant negative properties. Dots are colored according to mutation site as in panel A.