# Highlighting the Dystonic Phenotype Related to GNAO1

Fernando Acosta, Catherine Nowak, William G. Wilson, Dora Steel, Thomas Wirth, Giacomo Garone, Manju A. Kurian, Amélie Piton, Francisca Millan, Aida Telegrafi, et al.

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► To cite this version:

Fernando Acosta, Catherine Nowak, William G. Wilson, Dora Steel, Thomas Wirth, et al. Highlighting the Dystonic Phenotype Related to GNAO1. Movement Disorders, 2022, 37 (7), pp.1547-1554. 10.1002/mds.29074. hal-03780376

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# Highlighting the Dystonic Phenotype Related to GNAO1

Thomas Wirth, MD,¹,²,³* Giacomo Garone, MD,⁴,⁵

Manju A. Kurian, PhD,⁶ ID Amélie Piton, PhD,²,³,⁷

Francisca Millan, MD,⁸ Aida Telegrafi, MS,⁸

Nathalie Drouot, Msc,³ Gabrielle Rudolf, PhD,¹,²,³

Jamel Chelly, MD,²,³,⁷ Warren Marks, MD,⁹

Lydie Burglen, MD,¹⁰ Diane Demailly, MD,¹¹

Phillipe Coubes, MD,¹¹ Mayte Castro-Jimenez, MD,¹²

Sylvie Joriot, MD,¹³ Jamal Ghoumid, MD,¹⁴

Jérémie Belin, MD,¹⁵ Jean-Marc Faucheux, MD,¹⁶

Lubov Blumkin, MD,¹⁷ Mariam Hull, MD,¹⁸ ID

Mered Parnes, MD,¹⁸ ID Claudia Ravelli, MD,¹⁹

Gaëtan Poulen, MD,¹¹ Nadège Calmels, PharmD,²,³,⁷

Andrea H. Nemeth, MD,²⁰ Martin Smith, MD,²⁰

Angela Barnicoat, MD,²¹ Claire Ewenczyk, MD,²²,²³

Aurélie Mêneret, MD,²²,²³ ID Emmanuel Roze, MD,²²,²³

Boris Keren, MD,²²,²³ Cyril Mignot, MD,²²,²³

Christophe Beroud, MD,²⁴ Fernando Acosta, Jr, MD,⁹

Catherine Nowak, MD,²⁵ William G. Wilson, MD,²⁶

Dora Steel, BMBCh,⁶ Alessandro Capuano, MD,⁵

Marie Vidailhet, MD,²²,²³ Jean-Pierre Lin, MD,²⁷ ID

Christine Tranchant, MD,¹,²,³ Laura Cif, MD,¹¹ ID

Diane Doummar, MD,¹⁹ and Mathieu Anheim, MD¹,²,³

¹Département de Neurologie, Hôpital de Hautepierre, Hôpitaux Universitaires de Strasbourg, Strasbourg ²Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Strasbourg, France ³Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France ⁴University Hospital Pediatric Department, IRCCS Bambino Gesù Children's Hospital, University of Rome Tor Vergata, Rome, Italy ⁵Movement Disorders Clinic, Department of Neurosciences, Bambino Gesù Children's Hospital, Rome, Italy ⁶Molecular Neurosciences, Developmental Neurosciences, UCL Great Ormond Street Institute of Child Health, London, United Kingdom ⁷Laboratoire de diagnostic génétique,

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

*Correspondence to: Dr. Thomas Wirth, Département de Neurologie, Hôpital de Hautepierre, Hôpitaux Universitaires de Strasbourg, 1, avenue Molière, 67098 Strasbourg, France; E-mail: thomas.wirth@etu.unistra.fr

Thomas Wirth and Giacomo Garone contributed equally to this work and should be considered as co-first authors.

Laura Cif, Diane Doummar, and Mathieu Anheim contributed equally to this work and should be considered as co-last authors.

Relevant conflicts of interest/financial disclosures: None.

**Funding agencies:** T.W. was funded by a grant from the Revue Neurologique for this work. The study was partly supported by a grant provided by France Parkinson.

**Received:** 26 January 2022; **Revised:** 2 May 2022; **Accepted:** 5 May 2022

Published online 20 June 2022 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/mds.29074

Nouvel Hôpital Civil, Hôpitaux universitaires de Strasbourg, Strasbourg, France ⁸GeneDx, Gaithersburg, Maryland, USA ⁹Cook Children's Medical Centre, Fort Worth, Texas, USA ¹⁰Centre de Référence des Malformations et Maladies Congénitales du Cervelet, Département de Génétique et Embryologie Médicale, APHP, Hôpital Trousseau, Paris, France ¹¹Département de Neurochirurgie, Unité des Pathologies Cérébrales Résistantes, Unité de Recherche sur les Comportements et Mouvements Anormaux, Hôpital Gui de Chauliac, Centre Hospitalier Régional Montpellier, Montpellier, France ¹²Service de Neurologie, Department of Clinical Neurosciences, Lausanne University Hospital (CHUV) and University of Lausanne (UNIL), Lausanne, Switzerland ¹³Department of Paediatric Neurology, University Hospital of Lille, Lille, France ¹⁴Univ. Lille, ULR7364 RADEME, CHU Lille, Clinique de Génétique Guy Fontaine, Lille, France ¹⁵Service de neurologie, CHU Tours, Tours, France ¹⁶Service de neurologie, Hôpital d'Agen, Agen, France ¹⁷Pediatric Movement Disorders Clinic, Pediatric Neurology Unit, Wolfson Medical Center, Holon, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel ¹⁸Pediatric Movement Disorders Clinic, Blue Bird Circle Clinic for Pediatric Neurology, Section of Pediatric Neurology and Developmental Neuroscience, Texas Children's Hospital, Houston, Texas, USA ¹⁹Sorbonne Université, Service de Neuropédiatrie-Pathologie du développement, centre de référence neurogénétique, Hôpital Trousseau AP-HP.SU, FHU I2D2, Paris, France ²⁰Oxford University Hospitals National Health Service Foundation Trust and University of Oxford, Oxford, United Kingdom ²¹Department of Clinical Genetics, Great Ormond Street Hospital, London, United Kingdom ²²Sorbonne Université/Inserm U1127/ CNRS UMR 7225/Institut du Cerveau, Paris, France ²³Service de neurologie, Hôpital la Pitié Salpêtrière, Sorbonne Université, Paris, France ²⁴Aix Marseille Université, INSERM, MMG, Bioinformatics & Genetics, Marseille, France ²⁵The Feingold Center for Children, Division of Genetics and Genomics, Boston Children's Hospital, Boston, Massachusetts, USA ²⁶Department of Pediatrics, University of Virginia, Charlottesville, Virginia, USA ²⁷Children's Neurosciences Department, Evelina London Children's Hospital, Guy's and St Thomas NHS Foundation Trust, London, United Kingdom

**ABSTRACT:** **Background:** Most reported patients carrying GNAO1 mutations showed a severe phenotype characterized by early-onset epileptic encephalopathy and/or chorea.

**Objective:** The aim was to characterize the clinical and genetic features of patients with mild GNAO1-related phenotype with prominent movement disorders.

**Methods:** We included patients diagnosed with GNAO1-related movement disorders of delayed onset (>2 years). Patients experiencing either severe or profound intellectual disability or early-onset epileptic encephalopathy were excluded.

**Results:** Twenty-four patients and 1 asymptomatic subject were included. All patients showed dystonia as prominent movement disorder. Dystonia was focal in 1, segmental in 6, multifocal in 4, and generalized in 13. Six patients showed adolescence or adulthood-onset dystonia. Seven patients presented with parkinsonism and 3 with myoclonus. Dysarthria was observed in 19 patients. Mild and moderate ID were present in 10 and 2 patients, respectively.
**Conclusion:** We highlighted a mild GNAO1-related phenotype, including adolescent-onset dystonia, broadening the clinical spectrum of this condition. © 2022 The Authors. *Movement Disorders* published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

**Key Words:** dystonia; GNAO1; phenotypes; mutation

GNAO1 mutations have been associated with two phenotypes: a severe, early-infantile epileptic encephalopathy with burst-suppression (EIEE17, OMIM 615473¹) and a neurodevelopmental disorder with involuntary movements (NEDIM, OMIM 617493²⁻⁴), with or without seizures. GNAO1 encodes the α-subunit of a heterotrimeric guanine nucleotide-binding protein (Gαα), which is widely expressed in the central nervous system, playing an important role in signal transduction through AMPc metabolism in the striatum.²,⁵,⁶ As the number of reports increased, it became evident that GNAO1-related encephalopathies encompass a continuous spectrum of neurological syndromes featuring variable association of movement disorders, psychomotor delay, intellectual disability (ID), and different types of epilepsy.²,⁷ GNAO1-related movement disorder usually starts in infancy. Choreoathetosis is usually described with spontaneous or trigger-induced exacerbations, potentially leading to *status dystonicus*, as a hallmark of the disease.² Most patients reported so far showed a severe phenotype, with recurrent exacerbations and significant disability. However, in a few atypical, milder cases, with movement disorder onset in late childhood or adolescence, no acute exacerbation and less-severe disability have been identified using next-generation sequencing techniques.⁸⁻¹⁰ In this study, we characterized the clinical and genetic features of a cohort of patients with mild GNAO1-related phenotype characterized by prominent movement disorders, further expanding the spectrum of this condition.

# Patients and Methods

## Patients

Patients carrying causative heterozygous variants in GNAO1 and exhibiting mild phenotypes were included from 18 neurology and neuropediatric movement disorders reference centers from the United States, France, Israel, Switzerland, the United Kingdom, and Italy. Mild phenotype was defined by (1) lack of severe or profound ID, (2) lack of early-onset epileptic encephalopathy, (3) late-onset (ie, after age 2 years) appearance of movement disorders, and (4) acquisition of walk. Patients were

recruited through an international collaboration mediated by the online platform Genematcher.¹¹ All patients were assessed by neurologists or neuro-pediatricians with an expertise in movement disorders. Patients' phenotypes from family 6 and family 4, which were previously reported elsewhere, were added in the cohort as further clinical data were obtained.

## Genetic Analysis

CGH-array, gene panel, exome, and genome sequencing were performed as previously reported.⁹,¹⁰,¹²⁻¹⁵ Detailed procedures of the sequencing, including library preparation and bioinformatic analysis, are available in *Supplementary Data*. Variants were considered as causative if they fulfilled the following criteria: (1) known disease mutation reported in ClinVar; (2) loss-of-function variant, including protein truncating variants, frameshift indel, large deletion, and splice site changes predicted to cause aberrant splicing; or (3) missense variant with a CADD score >20, absent in GnomAD and predicted to be deleterious by at least two additional algorithms (Polyphen-2, SIFT and Mutation taster). In addition, variant class of pathogenicity was reported according to the American College of Medical Genetics and Genomics (ACMG) guidelines.¹⁶

## Ethics

All patients and relatives provided written informed consent before genetic analysis. Strasbourg University Hospital review board gave approval for the exome sequencing of families 4 and 6 that was performed in a research framework. Genetic analysis for other families was performed for diagnostic purposes.

## Results

We included 24 patients (15 women) and 1 asymptomatic carrier from 20 different families. Patients' clinical characteristics are provided in Table 1. Mean age at inclusion was 23.8 years (range: 5–66), mean age at disease onset was 6.6 years (range: 0.25–47), and mean age of dystonia onset was 10.1 years (range: 2–47). Initial manifestations included dystonia in 10 (41%), myoclonus or seizure in 1, developmental delay in 13, language delay in 4, motor delay in 9, and hypotonia in 4 patients. Seven patients were from three unrelated families showing autosomal dominant inheritance, while all others were sporadic cases due to de novo mutations. Pedigrees of these three families and videos of patients are available in *Supplementary Data*.

Dystonia was the main movement disorder in all patients, prominently affecting multiple segments of the upper body part in 21 patients or being limited to the cervical segment in 1 patient. Dystonia was isolated, namely not associated to any other symptom, in
<table><caption>TABLE 1 Clinical and genetic features of GNAO1 mutation carriers</caption><thead><tr><th rowspan="2">Patient ID</th><th rowspan="2">Ancestry</th><th rowspan="2">Gender</th><th rowspan="2">Age at last assessment</th><th rowspan="2">Age at first symptoms</th><th rowspan="2">First symptom</th><th colspan="2">Dystonia</th><th rowspan="2">Acute exacerbations</th><th rowspan="2">Parkinsonism</th><th rowspan="2">Myoclonus</th><th rowspan="2">Chorea</th><th rowspan="2">Hypotonia</th><th rowspan="2">Intellectual disability</th><th rowspan="2">Seizures</th><th rowspan="2">Speech</th><th rowspan="2">Other</th><th rowspan="2">Treatment response</th><th rowspan="2">GNAO1 variant</th></tr><tr><th>Dystonia age at onset</th><th>Topography</th></tr></thead><tbody><tr><td>Family 1<br>Case A</td><td>North African</td><td>Female</td><td>28 y</td><td>4 y</td><td>Seizure</td><td>12 y</td><td>Segmental face, neck, upper limbs</td><td>No</td><td>No</td><td>Akistic-idal syndrome</td><td>No</td><td>No</td><td>No</td><td>Mild</td><td>Yes</td><td>Normal</td><td>None</td><td>Mild response to levodopa, moderate improvement with trizephenyl</td><td>[NML_020988_3]; c.68 T &gt; C; p.L29P; ltr</td></tr><tr><td>Family 2<br>Case A</td><td>North African</td><td>Female</td><td>5 y</td><td>By 1 y</td><td>Developmental delay (inotor delay)</td><td>2 y</td><td>Generalized: oromandibular, trink. Dysonic gas</td><td>No</td><td>No</td><td></td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>Dyarthria</td><td>None</td><td>NA</td><td>[NML_020988_3]; c.137A &gt; G; p.K40R; ltr</td></tr><tr><td>Family 3<br>Case A</td><td>European</td><td>Male</td><td>19 y</td><td>3 mo</td><td>Developmental delay (inotor delay, hypotonia)</td><td>12 y</td><td>Generalized: left upper limb, cervical and axial dysonia, BFMDRS: 16</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>Mild</td><td>Yes</td><td>Dyarthria</td><td>None</td><td>No response to levodopa and tetraphenazine, minimal improvement with geipapetin and trifluphenyl</td><td>[NML_020988_3]; c.535A &gt; G; p.R179G; ltr</td></tr><tr><td>Family 4<br>Case A</td><td>European</td><td>Male</td><td>24 y</td><td>15 y</td><td>Dystonia</td><td>15 y</td><td>Segmental: oromandibular and cervical dysonia</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Dyarthria</td><td>None</td><td>No response to levodopa and tetraphenazine, mild worsening by Gpi-DBS</td><td>[NML_020988_3]; c.617G &gt; A; p.R260Q; ltr</td></tr><tr><td>Family 4<br>Case B</td><td>European</td><td>Female</td><td>53 y</td><td>47 y</td><td>Dystonia</td><td>47 y</td><td>Focal: cervical</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Normal</td><td>None</td><td>NA</td><td></td></tr><tr><td>Family 4<br>Case C</td><td>European</td><td>Female</td><td>57 y</td><td>30 y</td><td>Dystonia</td><td>30 y</td><td>Multifocal: upper and lower limbs, laryngeal dysonia with dyarthria</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Dyarthria</td><td>None</td><td>NA</td><td></td></tr><tr><td>Family 5<br>Case A</td><td>European</td><td>Female</td><td>5 y 11 mo</td><td>3 mo</td><td>Developmental delay (inotor delay, hypotonia)</td><td>5 y</td><td>Multifocal: four limbs</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>Dyarthria language delay</td><td>None</td><td>NA</td><td>[NML_020988_3]; c.620G &gt; C; p.E288N; ltr</td></tr><tr><td>Family 6<br>Case A</td><td>European</td><td>Male</td><td>31 y</td><td>3 y</td><td>Dystonia</td><td>3 y</td><td>Generalized: left lower limb dysonia, bilateral upper limbs dysonia, laryngeal dysonia, abnormal axial posture, BFMDRS: 24.5</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>Mild</td><td>No</td><td>Dyarthria</td><td>None</td><td>Mild response to levodopa, mild response to trizephenyl</td><td>[NML_020988_3]; c.644G &gt; A; p.C215Y; ltr</td></tr><tr><td>Family 6<br>Case B</td><td>European</td><td>Female</td><td>66 y</td><td>5 y</td><td>Dystonia</td><td>5 y</td><td>Generalized: laryngeal dysonia, facial dysonia, axial dysonia, abnormal posture of the left hand and bilateral pre-vulva BFMDRS: 23.5</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Dyarthria</td><td>Pyramidal syndrome</td><td>Subjective response to levodopa</td><td></td></tr><tr><td>Family 7<br>Case A</td><td>European</td><td>Male</td><td>48 y</td><td>6 y</td><td>Dystonia</td><td>6 y</td><td>Generalized: oromandibular, cervical, trink. upper limbs, left foot</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Mild</td><td>No</td><td>Dyarthria</td><td>None</td><td>Subjective response to levodopa: no effect of trizephenyl</td><td></td></tr><tr><td>Family 8<br>Case A</td><td>European</td><td>Female</td><td>16 y</td><td>6 y</td><td>Dystonia</td><td>6 y</td><td>Segmental: oropharyngeal, neck and trapezius</td><td>Yes</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Mild</td><td>No</td><td>Severe dysarthria</td><td>MDI</td><td>Mild improvement with geipapetin, no benefit with trizephenyl</td><td></td></tr></tbody></table>

(Continues)
<table><caption>TABLE 1 Continued</caption><thead><tr><th rowspan="2">Patient ID</th><th rowspan="2">Ancestry</th><th rowspan="2">Gender</th><th rowspan="2">Age at last assessment</th><th rowspan="2">Age at first symptoms</th><th rowspan="2">First symptom</th><th colspan="2">Dystonia</th><th rowspan="2">Myoclonus</th><th rowspan="2">Chores</th><th rowspan="2">Hypotonia</th><th rowspan="2">Intellectual disability</th><th rowspan="2">Seizures</th><th rowspan="2">Speech</th><th rowspan="2">Other</th><th rowspan="2">Treatment response</th><th rowspan="2">GN401 variant</th></tr><tr><th>Dystonia age of onset</th><th>Topography</th><th>Progression</th><th>Acute exacerbations</th></tr></thead><tbody><tr><td>Family 9<br>Case A</td><td>African American</td><td>Male</td><td>13 y</td><td>7 mo</td><td>Developmental delay (language delay)</td><td>5 y</td><td>Generalized initially axial (opithecotonic with neck involvement) with secondary limbs and oromandibular involvement</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Dyarthria</td><td>None</td><td>No response to levodopa and trihexaphenyl; rath with clozapine; minimal improvement with baclofen</td><td>[NML_020988-3]; c.724_8G > A, hz</td></tr><tr><td>Family 10<br>Case A<br>Jewish</td><td>Moroccan Syrian</td><td>Female</td><td>15 y</td><td>1 y</td><td>Developmental delay (motor delay)</td><td>5 y</td><td>Generalized dystonia, BFMDRS: 48.5</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Dyarthria</td><td>Exaggerated startle reflex</td><td>No response to levodopa</td><td></td></tr><tr><td>Family 11<br>Case A</td><td>European</td><td>Female</td><td>18 y 6 mo</td><td>By 1 y</td><td>Developmental delay (motor delay)</td><td>7 y</td><td>Generalized cervical extension, dystonic forward trunk, BFMDRS: 85</td><td>Yes</td><td>No</td><td>Severe akinetic rigid syndrome</td><td>No</td><td>No</td><td>Mild</td><td>No</td><td>Dyarthria</td><td></td></tr><tr><td>Family 12<br>Case A</td><td>European</td><td>Female</td><td>21 y</td><td>By 1 y</td><td>Developmental delay (motor delay)</td><td>7 y</td><td>Generalized dystonia with severe cervical neck extension and oromandibular dystonia, BFMDRS: 64.5</td><td>Yes</td><td>No</td><td>Akinetic rigidly facial akinesia</td><td>No</td><td>No</td><td>Yes</td><td>Mild</td><td>Anathira</td><td></td></tr><tr><td>Family 13<br>Case A</td><td>Caucasian</td><td>Male</td><td>20 y 2 mo</td><td>By 1 y</td><td>Developmental delay (motor delay, language delay)</td><td>11 y</td><td>Generalized bilateral upper limb, axial, trunk, cervical, oromandib- pharyngodarynx dysonia with speech and swallowing impairment; dysonic gait, BFMDRS: 45.5</td><td>Yes</td><td>Yes</td><td>Akinetic-rigid syndrome</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>Severe dysarthria</td><td></td></tr><tr><td>Family 14<br>Case A<br>Chinese European</td><td>Male</td><td>13 y</td><td>3 y</td><td>Developmental delay (language delay) myoclonus</td><td>11 y</td><td>Segmental bilateral upper limb, dysonia</td><td>No</td><td>No</td><td>No</td><td>Yes (upper limbs)</td><td>No</td><td>Yes</td><td>Mild</td><td>No</td><td>Normal</td><td>ASD</td><td>No response to acetazolamide and amantadine; levodopa; moderate and transient response to methylphenidate and trihexaphenyl; good response to Gp-DBS</td></tr><tr><td>Family 15<br>Case A<br>Mixed European</td><td>Female</td><td>11 y 8 mo</td><td>10 mo</td><td>Developmental delay</td><td>2 y</td><td>Generalized upper and lower limbs, axial, dysonia gait</td><td>Yes</td><td>No</td><td>No</td><td>Yes (upper limbs)</td><td>No</td><td>Yes (left side)</td><td>Yes</td><td>No</td><td>No</td><td>Dyarthria</td><td>ADHD</td><td>Moderate response to terterabenzone on chorea, good response to trihexaphenyl</td></tr><tr><td>Family 16<br>Case A<br>Northern European</td><td>Female</td><td>5 y 6 mo</td><td>3 mo</td><td>Developmental delay (motor delay, hypotonia)</td><td>5 y</td><td>Segmental, dystonic posturing of fingers and hands</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>Mild</td><td>No</td><td>Dyarthria</td><td>None</td><td>Good response to levodopa</td></tr><tr><td>Family 17<br>Case A</td><td>Caucasian</td><td>Male</td><td>18 y</td><td>2 y</td><td>Developmental delay (language delay)</td><td>2 y</td><td>Segmental, laryngeal, right upper limb (handwriting) and cervical</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Dyarthria</td><td>None</td><td>Response to anticholinergic and levodopa</td></tr><tr><td>Family 18<br>Case A</td><td>European</td><td>Female</td><td>20 y 4 mo</td><td>6 y</td><td>Dystonia</td><td>6 y</td><td>Generalized dystonia, BFMDRS: 67.5</td><td>Yes</td><td>Yes</td><td>Bradykinesia-akinesia</td><td>No</td><td>Yes generalized</td><td>Yes</td><td>Mild</td><td>No</td><td>Anathira</td><td>None</td><td>No response to haloperidol, amantadine; and trihexaphenyl</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 1 Continued</caption><thead><tr><th rowspan="2">Patient ID</th><th rowspan="2">Ancestry</th><th rowspan="2">Gender</th><th rowspan="2">Age at last assessment</th><th rowspan="2">Age at first symptoms</th><th rowspan="2">First symptom</th><th colspan="2">Dystonia</th><th rowspan="2">Acute exacerbations</th><th rowspan="2">Parkinsonism</th><th rowspan="2">Myoclonus</th><th rowspan="2">Chorea</th><th rowspan="2">Hypotonia</th><th rowspan="2">Intellectual disability</th><th rowspan="2">Seizures</th><th rowspan="2">Speech</th><th rowspan="2">Other</th><th rowspan="2">Treatment response</th><th rowspan="2">GNAQI variant</th></tr><tr><th>Dystonia age of onset</th><th>Topography</th><th>Progression</th><th>Exacerbations</th></tr></thead><tbody><tr><td>Family 19<br>Case A</td><td>Female</td><td>13 y</td><td>6 y</td><td>Dystonia</td><td>6 y</td><td>Multifocal bilateral upper limb, cervical, and oromandibular dystonia</td><td>No</td><td>No</td><td>Mild atkinetic rigid syndrome</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>Dysarthria</td><td>None</td><td>NA</td><td>[NNM_020988_3]; c.76dup; T; p.N256*; hz</td></tr><tr><td>Family 19<br>Case B</td><td>Female</td><td>39 y</td><td>16 y</td><td>Dystonia</td><td>16 y</td><td>Multifocal bilateral upper limb, cervical, and oromandibular dystonia</td><td>No</td><td>No</td><td>Mild atkinetic rigid syndrome</td><td>No</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Dysarthria</td><td>None</td><td>No response to clonazepam; sustained response to Gpi-DBS</td><td>Heterozygous deletion in levodopa, clonazepam, or baclofen (273-375 kb) encompassing GNAQI</td></tr><tr><td>Family 20<br>Case A</td><td>Canaaan</td><td>Male</td><td>9 y</td><td>By 1 y</td><td>Developmental delay (notor delay, hypotonia)</td><td>9 y</td><td>Generalized upper-limb and trunk dystonia, dystonic gait</td><td>Yes</td><td>No</td><td>No</td><td>No</td><td>No</td><td>Yes</td><td>No</td><td>No</td><td>Normal</td><td>None</td><td>No response to levodopa, clonazepam, or baclofen</td><td>syndrome: 1<br>MDI: 1<br>ADHD: 2<br>ASD: 1<br>Exaggerated startle reflex: 1</td></tr><tr><td>Summary</td><td>Female<br>15<br>Male 9</td><td>Mean age<br>at last<br>assessment:<br>23.8 y</td><td>Mean age<br>at<br>disease<br> onset:<br>6.6 y</td><td>Developmental<br>delay: 13<br>Motor delay: 9<br>Language delay: 4<br>Dystonia: 10<br>Hypotonia: 4<br>Seizure: 1<br>Myoclonus: 1</td><td>Mean age at<br>dystonia<br>onset:<br>10.1 y</td><td>Focal:<br>Segmental: 6<br>Multifocal: 4<br>Generalized: 13</td><td>Progression<br>13</td><td>Exacerbation<br>3</td><td>Parkinsonism<br>7</td><td>Myoclonus<br>3</td><td>Chorea<br>2</td><td>Hypotonia<br>11</td><td>Intellectual<br>disability<br>12</td><td>Seizures<br>3</td><td>Dysarthria:<br>19</td><td>Pyramidal</td><td>syndrome: 1<br>MDI: 1<br>ADHD: 2<br>ASD: 1<br>Exaggerated startle reflex: 1</td><td></td></tr></tbody></table>

Abbreviations: htz: heterozygous, NA, not available; BFMDRS: Burke Fahn Marsden Dystonia Rating Scale, dystonia score; GPi-DBS; globus pallidus internal deep brain stimulation; MDI, major depressive disorder; ADHD, attention deficit with hyperactivity disorder; ASD, autism spectrum disorder; ID, intellectual disability.
7 patients (29%). Dystonia was the only movement disorder in 14 patients and was combined with other movement disorders in 10, namely myoclonus in 3, chorea in 2, and parkinsonism in 7 (with 2 patients combining three movement disorders). Only 3 patients presented an acute exacerbation of dystonia. Dystonia course was nonprogressive for 11 patients. Dystonia topography was generalized in 13 patients (54%), multifocal in 4 patients, segmental in 6 patients, and focal in 1 patient. Dystonia was associated with dysarthria/anarthria in 19 patients. Early-onset hypotonia preceded dystonia in 11 patients. Dystonia was associated with ID in 12 patients (mild for 10 and moderate for 2). Seizures occurred in 3 patients between age 4 and 19 years. Magnetic resonance imaging was unremarkable for all patients.

Movement disorders response to medication, including anticholinergic drugs, levodopa, tetrabenazine, amantadine, clonazepam, or methylphenidate, was variable. Six patients received pallidal deep brain stimulation (DBS), with significant improvement for 5 of them. Detailed treatment outcomes are available in *Supplementary Data*.

Mutations carried by the patients are presented in Table 1. Details regarding pathogenicity assessment are available in *Supplementary Data*. Apart from the p.R206Q, all variants were classified as pathogenic (class V) according to the ACMG criteria. In family 4, we identified 3 patients with the R206Q variant, which was classified as a variant of uncertain significance due to the presence of an unaffected carrier, 4-D, son of 4-C, despite meeting other criteria of pathogenicity (absent from GnomAD, unanimously predicted damaging by in silico tools, affecting a highly conserved residue located in a hot spot without benign variation) (criteria PM1, PM2, PP2, and PP3). A recurring splicing variant (c.724-8G > A), previously reported in ClinVar, was identified in 8 patients (33%) showing late-onset and/or segmental dystonia. Previous report showed this variant to damage the natural splice acceptor site and create a stronger cryptic splice acceptor site in intron 6, resulting in the insertion of two amino acids leading to protein mislocation.¹⁷ Two patients were carrying a nonsense variant (p.N256*), and one was carrying a

<figure><img src="image_3.png" /><figcaption>**FIG. 1.** Impact of the mutations on the protein. (A) Position of the variant sites on the heterotrimeric complex containing the Gα subunit. The heterotrimer is depicted in the resting state (GDP-bound, PDBcode 1GG2). Subunits α, β, and γ are colored in green, cyan, and gray, respectively. Affected residues in this cohort are in red, and their position is indicated both on the human Gαo1 and on rat Gαi1 (UniProtKB ID P10824, between brackets). GDP-binding residues are colored in yellow. Previously reported GNAO1 variants are in blue. On the right, a focused view of the GDP-binding site is shown. (B) Cartoon model of the heterotrimeric-αβγ G-protein coupled-receptor on the synaptic cleft. (C) Schematic representation of the disease-causing variants on GNAO1 transcript (NM_020988.3) and protein (UniprotKB ID P09471-1). The amino acids impacted by the mutations identified in this work are in red, whereas previously reported variants are in black. The blue bar on the transcript indicates the translated region. The blue segments in the protein sequence indicate the G-motifs (containing the nucleotide binding residues)—numbered from 1 to 5. Molecular graphics are realized with UCSF Chimera (http://www.rbvi.ucsf.edu/chimera), developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311. The cartoon has been created with BioRender.com. aa, amino acids; nt, nucleotides; UTR, untranslated region. [Color figure can be viewed at wileyonlinelibrary.com]</figcaption></figure>
large deletion encompassing GNAO1. Other mutations (p.L23P; p.K46R; p.R179G; p.R206Q; p.E208N; p.C215Y; and p.N242T and p.E246V) were all missense variants absent from GnomAD. The previously reported pathogenic p.C215Y variant⁸ was found in three unrelated families. All variants were close to known mutational hot spots (Fig. 1).

Discussion

Here, we report a large cohort of patients with mild GNAO1-related phenotypes, experiencing prominent movement disorders without severe chronic encephalopathy. The typical phenotype was a nonprogressive generalized or focal/segmental upper-body dystonia appearing beyond infancy, associated with dysarthria. Acute exacerbation occurred only in 3 patients, and 29% of patients showed isolated dystonia without additional neurological manifestation. Our inclusion criteria were able to identify these phenotypes that were in contrast with most of the previously reported patients with GNAO1-related movement disorders, who showed severe hyperkinetic encephalopathy with recurrent dystonic exacerbations,¹⁸,¹⁹ and profound developmental delay³,⁷ with or without epilepsy in the first year of life.¹,⁷

Dystonia distribution was segmental or focal in 7 patients, and clinical course was nonprogressive in 11 patients, while most of the previously reported patients with GNAO1-related movement disorders had generalized and rapidly progressive dystonia.² Dystonia topography revealed prominent upper-body distribution in most of our patients, reminiscent of the clinical pictures associated with other dystonia-related genes, such as GNAL²⁰,²¹ or ANO3.²²,²³ Seven patients also exhibited mild parkinsonism, which is consistent with the role of Gαα in the signal transduction within the striatal projection neurons downstream of the dopamine receptors.⁶,²⁴

We identified 3 autosomal dominant families where multiple symptomatic relatives carried heterozygous variants, which was in contrast with all the previously reported patients who showed de novo mutations.² One p.R206Q carrier did not present any clinical sign evocative of GNAO1-related disorders. The similarities between this family's phenotype and the other cases— all showing upper-body distribution—argue for the implication of the variant, while no other class IV to V variant in a dystonia-related gene was identified. In addition, a family member carrying this variant had disease onset in his 40s, meaning the 30-year-old asymptomatic carrier could be potentially presymptomatic. Future identification of autosomal dominant family with GNAO1-related dystonia and follow-up of this patient might confirm whether incomplete penetrance is possible in GNAO1-related disorders.

Response to medication was variable in our cohort. No significant response to levodopa was identified in our cohort, but 3 patients had partial response to anticholinergic drugs, which was in accordance with previous findings from the literature.²,⁴,²⁵ Conversely, the outcome was good in 5 of 6 patients who received DBS, further confirming its efficacy in GNAO1-related dystonia.²⁶

Most of the variants identified in the present work were not reported among previously published cases showing severe phenotype, and two variants recurred in multiple families, suggesting that mild phenotypes could be related to specific mutations. However, the variants we identified were close to previously reported hot spots (Fig. 1), leading to amino-acid substitution in the same functional domains. Further studies are needed to elucidate if these different variants have a milder impact on protein function. In addition, we identified two putative loss-of-function variants (a nonsense variant and a whole-gene deletion), presumably affecting protein expression and possibly causing GNAO1 haploinsufficiency. All 3 carriers were presenting late-childhood/adolescence onset dystonia without ID. Thus far, no report described the phenotype of patients harboring GNAO1-nonsense variants. A few patients with chromosome 16q deletions encompassing GNAO1 have been described, all harboring significantly larger deletions compared to our case and showing variable associations of dysmorphisms, microcephaly, seizures, and developmental delay.²⁷ Although previous research suggested that loss-of-function variants were mainly responsible for epileptic encephalopathy while gain-of-function mutations were mostly associated with a movement disorders prominent phenotype,⁵ recent evidence suggests that pathogenic variants exert their effect through a combination of dominant-negative and loss-of-function mechanisms, and each mutation likely produces circuit-selective effects through a peculiar mechanism of signaling disruption.²⁸ The expanding spectrum of associated phenotypes and disease-causing variants provides further evidence that genotype-phenotype correlations are nuanced, and GNAO1-related disorders shape a continuous spectrum of overlapping phenotypes rather than distinct entities.²⁸ Our study carries some limitations, including the retrospective design and the lack of formal assessment in several cases. Here, we highlighted the milder GNAO1-related phenotypes, broadening this condition-clinical spectrum. GNAO1 mutations should be considered as a cause of adolescent or adult-onset nonprogressive dystonia, particularly in the presence of a speech involvement even in the absence of seizures or ID. ■

**Acknowledgments:** We thank the patients and their families for participation in this study and Bernard Jost from the IGBMC Microarray and Sequencing Platform, the France Parkinson organization, and the *Revue Neurologique* for their support. Open access funding enabled and organized by Projekt DEAL.
# Data Availability Statement

Anonymized data pertaining to the research presented will be made available upon reasonable request from external investigators.

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# Supporting Data

Additional Supporting Information may be found in the online version of this article at the publisher's web-site.