Diverse faces of GNAO1: Mild forms in epilepsy and autism

Dear Sirs,

GNAO1-related disorders (GNAO1-RD) are caused by variants in the GNAO1 gene, encoding the Gαo protein. GNAO1-RD patients typically present with moderate to severe symptoms, such as epilepsy, movement disorders, developmental delay (DD) or intellectual disability (ID), and the need for a gastrostomy.¹ Approximately 50% of GNAO1-RD patients exhibit epileptic manifestations. In two-thirds of these cases, epilepsy manifests as DEE, especially if it begins within the first year of life. The remaining 30% may, at some point, develop epilepsy². Herein, we report two cases of exceptionally mild GNAO1-RD phenotypes, underscoring the importance of investigating mutations in GNAO1 in patients with a mild neurological phenotype.

Patients from the ongoing study “Prospective and retrospective study of phenotypic and genotypic characterization of patients affected by GNAO1-RD (PIC-150–23) at the Hospital Sant Joan de Déu, Barcelona, Spain, as well as patients from neurologists in Europe who collaborated on this research, were included in the study. Clinical records were retrospectively reviewed for study data. Patients were assessed using the GNAO1-RD Severity Score ¹. The Supplementary material describes C215F functional characterization methods.

Milder GNAO1 cases presenting with epilepsy, ASD, and ADHD (Table 1)

Table 1.

Overview of Clinical, Genetic, and Radiological Features

	P1	P2
Ethnic background	Spanish	Italian
Sex	Male	Male
GNAO1 variant (NM_020988.3)	c.412C>T/p.E138*	c.644G>A/p.C215F
Age at last exam (years)	14	14
Age at sitting (months)	9	6
Age at walking (months)	15	12
Age at first words (years)	1	1
Intellectual disability	ASD, ADHD	Subtle disfluency noted at 4 years of age.
Movement disorders	Mild left foot dystonia	Subtle postural tremor
Seizures (age onset)	Simple febrile seizure at 14 months. Motor generalized onset seizure with fever at 6.5 years of age	Motor and nonmotor generalized onset seizures at 7 years of age
VideoEEG	Normal background activity. Multifocal bilateral epileptiform abnormalities during sleep.	Normal awake and sleep EEG
Treatment	Valproic acid, lisdexamfetamine dimesylate	Valproic acid
Neuroimaging (MRI)	Normal	Normal
GNAO1 Severity Score
Epilepsy	0,25	0,25
Movement Disorders	0,25	0,25
Gross motor development	0	0
Language development	0	0
Feeding	0	0
Total	0,5	0,5

Autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD)

Patient 1, a 14-year-old Spanish male, was born at full term. He displayed a mild motor delay, achieving the ability to sit independently at 9–10 months of age and walk unassisted at 15 months. He exhibited impaired social interaction, literal speech, and attention difficulties, leading to the diagnosis of ASD and attention deficit hyperactivity disorder (ADHD). After the adverse effects of methylphenidate and guanfacine, lisdexamfetamine dimesylate improved his sustained attention. Despite these challenges, he attends regular school with time-management-related methodological changes. After a simple febrile seizure at 14 months, he presented with epilepsy at 6.5 years, with subsequent episodes occurring over the next two years. These first seizures were described as bilateral tonic-clonic seizures of unknown onset. Valproic acid was started after the second seizure, with a good response, so after a 4-year period of seizure freedom, treatment was withdrawn. At 13, he had a focal-onset seizure with impaired awareness, with some weird feeling that the patient cannot explain, head and eye deviation, and loss of consciousness. He is currently under treatment with valproic acid (Supplemental Data). Physical examination revealed minimal left foot distal dystonia (Video S1). The GNAO1-RD severity score was 0.5 (mild). A brain MRI revealed no abnormalities. Trio-Whole Genome Sequencing (WGS) identified a de novo GNAO1 variant (NM_020988.3): c.412C>T/p.E138*.

Patient 2, a 14-year-old Italian male, was born at term. Early neurodevelopment was typical, but at the age of 4, he started speech therapy due to a slight dysfluency. At 7 years old, he experienced generalized absence seizures characterized by EEG findings of typical 3-Hz generalized spike and wave discharges. Valproic acid controlled seizures, but two years later, withdrawal caused a generalized tonic-clonic seizure, requiring medication reinstatement. A brain MRI at 7 years old was normal. Neuropsychological assessment at age 13 indicated a mild cognitive deficit, including deficits in attention, verbal memory, and reasoning (Supplemental Data). Neurological examination was normal except for a subtle postural tremor (Video S1). The GNAO1-RD severity score was 0.5 (mild). The epilepsy gene panel revealed a de novo mutation in the GNAO1 gene (NM_020988.3):c.644G>A/p.C215F.

The American College of Medical Genetics classifies E138* and C215F variants as pathogenic, with CADD scores of 41.4 and 29.4, respectively.

Impaired dopamine signaling and an altered G-protein interaction pattern are due to the novel GNAO1 C215F variant.

C215 is located in the switch II region pivotal for interactions of Gαo with its effectors and Gβγ (Figure 1A). We observed no significant difference between WT and C215F Gαo expression levels (Figure 1B). Using bioluminescence resonance energy transfer (BRET) (Figure 1C), we found that, after dopamine stimulation, the C215F variant displayed a significantly reduced maximum response as compared to WT (Figure 1D, E). The C215F variant had no effect on G protein heterotrimer formation (Figure 1F). Previously, we reported that several Gαo variants display dominant negative activity³. We found that the C215F variant displayed a modest but significant decrease in dopamine response, indicating that it interferes with normal activation of Gαo by D2R (Figure 1G, H, I). The C215F variant caused a significant increase in interaction between the receptor and Gβγ compared to WT. This suggests that C215F may increase heterotrimer recruitment upon agonist stimulation and/or reduce Gαo and Gβγ release upon GPCR activation (Figure 1J, K).

Figure 1.

GNAO1 C215F variant exhibits an inhibited agonist response.

A. Location of C215 on Gαo (PDB: 3C7K). Regions are indicated as follows: Purple – P-loop; Cornflower blue – Switch I; Cyan – Switch II; Blue – Switch III; Yellow – C215.

B. Expression level of GNAO1 C215F variant. HEK293FT cells were transfected with WT or C215F Gαo or pcDNA control, lysed and then immunoblotted with α-Gαo antibody.

C. Schematic of the G protein coupled receptor (GPCR) signaling assay. GPCR activation allows Gαo to release venus-tagged Gβγ which then interacts with NanoLuc-tagged GRK, causing a shift in the BRET ratio.

D. Representative traces of dopamine BRET responses.

E. The effect of GNAO1 C215F variant on dopamine induced BRET signal. The maximum amplitude of the change in BRET signal was measured in comparison to the average baseline BRET value.

F. The effect of GNAO1 C215F variant on basal BRET. 0% heterotrimer formation was defined as the amount of basal BRET without Gαo expressed. The degree of heterotrimer formation was calculated by subtracting the basal BRET value of WT or C215F Gαo expressing cells from the Gαo-free control. The results are normalized to the WT Gαo control.

G. Representative traces of dopamine BRET responses of the GNAO1 C215F variant expressed alongside WT Gαo.

H. The effect of the GNAO1 C215F variant on WT Gαo dopamine induced BRET signal. 100% activity was defined as the maximum amplitude of condition expressing pcDNA alongside WT Gαo (WT: Control). The maximum amplitude of WT: C215F condition falls below this value, indicating that the variant has dominant negative activity. The assay is saturated with WT Gαo, as indicated by the non-significant difference with the condition expressing 2x WT Gαo (WT: WT).

I. Schematic of the receptor interaction assay. A luminescent readout is given after the SmBiT tag on D2R interacts with the LgBiT tag on Gβ and reconstitutes NanoLuc.

J. Representative luminescence traces of the interaction of G protein heterotrimer with D2R receptor.

K. The effect of the GNAO1 C215F variant on dopamine-induced receptor interaction with G protein heterotrimer. Runs were normalized by taking the amount of luminescence detected after stimulation of the Gαo-free control with dopamine as 0% interaction and the amount of luminescence detected with WT Gαo as 100% interaction.

This study presents two patients with novel GNAO1 variants, E138* and C215F, who have mild neurological phenotypes with epilepsy, ASD, ADHD, and mild movement disorders (focal dystonia and postural tremor). These patients lack the typical moderate to severe DD/ID associated with GNAO1-RD, thereby expanding the known spectrum of GNAO1-RD. Our findings underscore the significance of investigating GNAO1 gene variants, even in cases with mild neurological manifestations.

The newly identified C215F variant shares similarities with variants like C215Y in Gαo, which present a mild pathology. All five C215Y cases had early-onset generalized dystonia, speech impairment, and hypotonia, mildly affecting psychomotor development. These cases did not show epilepsy ^4,5.

Studies involving mice with the C215Y variant have shown altered behavior and motor abnormalities resembling human symptoms,⁶ without epilepsy or developmental delays. However, our study revealed that C215F does not impact basal Gαo-Gβγ interaction but moderately impedes Gβγ release upon GPCR activation. The C215F mutant, despite exhibiting moderate dominant negative activity and reduced agonist-induced activation, did not impact heterotrimer formation and was associated with mild clinical severity. This further reinforces the idea that heterotrimer assembly and controlling Gβγ signaling at the basal state are the key factors that contribute to the severity of the clinical phenotype in GNAO1-RD.

Regarding the E138* variant, a recent study by Galosi et al. described an 8-year-old boy exhibiting subtle neurological manifestations,⁷ including generalized tonic–clonic seizures during fever, mild language impairment, dystonic postures of the lower limbs during walking, and occasional tongue dyskinetic movements. This patient carried the NM_020988.3:c.163_164del/I55H*3 variant, leading to nonsense-mediated decay of the transcript and resulting loss of protein. Considering the earlier termination caused by the E138* variant, we suggest a comparable mechanism resulting in the complete elimination of mutant Gαo, leading to haploinsufficiency and contributing to the disease pathology in the affected patient. It is worth noting that not all cases of haploinsufficiency result in mild phenotypes, as observed in patients with I55H*3⁷, N256*⁵, and a 16q12.2q21 deletion, including the whole GNAO1 gene deletion.

In summary, we found that the GNAO1 variant C215F has a distinct impairment in Gαo functionality, resulting in an inability to release Gβγ in response to agonist stimulation during heterotrimer formation. This novel insight emphasizes the need to thoroughly study GNAO1 gene variants, including cases with mild neurological symptoms.

Supplementary Material

Figure S1

Figure S1. The GNAO1 C215F variant disrupts a network of van der Waals interactions.A. The location of residues interacting within 3–5Å of cysteine 215 (PDB: 3C7K). Regions are indicated as follows: Purple – P-loop; Cornflower blue – Switch I; Cyan – Switch II; Blue – Switch III; Yellow – C215; Orange red – β1 strand.B. Alignment of representative Gα subtype sequences, revealing that the residues interacting with C215 are conserved.C. There are no steric clashes with any allowed rotamer of cysteine 215.D. All allowed rotamers of phenylalanine 215 result in several steric clashes with neighboring residues. Clashes are identified by dashed purple lines.

Figure S2

Figure S2. Evolution of epileptic seizures, antiepileptic medication, and video EEG results in P1 and P2 with mild phenotype GNAO1-RD.

Supplemental Data_14_01_2024

Table S1. List of clashes caused by C215F rotamers.

Table S2. Neuropsychological assessment of GNAO1-RD patients.

Table S3. Genetics variants included in this study

MovieS13

Movie S1. Morph between the different nucleotide states of Gαi1. A close-up of the pocket of residues C214 interacts with in the GTP-bound state. As GTP is hydrolyzed, C214 rotates outward to bind to Gβ. GDP-bound Gαi1 with Gβγ, PDB:1GP2; Receptor activated Gαi1 (no nucleotide) with Gβγ, PDB:6KPF; GTP-bound Gαi1, PDB:1GIL. Regions are indicated as follows: Cornflower blue – Switch I; Cyan – Switch II; Blue – Switch III; Yellow – C215; Orange red – β1 strand; Orange – Receptor; Purple – Gβ; Green – Gγ.

Video 1

Video S1. Patient 1. In rest, dystonic posture of the left big toe is observed. No other movement disorders are noted. Patient 2. With hands extended forward and finger-to-nose test, no dystonia or associated chorea is observed. Tremor not visualized in the video.

Data availability.

All relevant data can be found in the article. The participant of this study did not give written consent for further data to be shared publicly, so due to the sensitive nature of the research further supporting data is not available.