Spectrum of Movement Disorders in GNAO1 Encephalopathy: in-depth Phenotyping and Literature Review


 Background

GNAO1 encephalopathy is a rare neurodevelopmental disorder characterized by distinct movement presentations and early onset epileptic encephalopathy. Here, we report the in-depth phenotyping of genetically confirmed patients with GNAO1 encephalopathy, focusing on movement presentations.
Results

Six patients who participated in Korean Undiagnosed Disease Program were diagnosed to have pathogenic or likely pathogenic variants in GNAO1 using whole exome sequencing. All medical records and personal video clips were analyzed with a literature review. Three of the 6 patients were male. Mean follow-up duration was 39 months (range, 7–78 months) and age at last examination was 8.0 years (range, 3.3–16.9 years). Initial complaints were hypotonia or developmental delay in 5 and right-hand clumsiness in 1 patient, which were noticed at 20 months of age on average (range, 0–75 months). All patients showed global developmental delay and 4 had severely retarded development. Five patients (5/6, 83.3%) had many different movement symptoms with various onset and progression. The symptoms included stereotyped hands movement, non-epileptic myoclonus, dyskinesia, dystonia and choreoathetosis. Whole exome sequencing identified 6 different variants in GNAO1. Three were novel de novo variants and atypical presentation was noted in a patient. One variant turned out to be inherited from patient’s mother who had mosaic variant. Distinct phenotypes in patients with variant p.Glu246Lys and p.Arg209His were elucidated by in-depth phenotyping and literature review.
Conclusions

We reported 6 patients with GNAO1 encephalopathy showing an extremely diverse clinical spectrum on video. Some characteristic movement features identified by careful inspection may also provide important diagnostic insight and practice guidelines.


Introduction
Since the rst identi cation of GNAO1 as a new causative gene of early-onset epileptic encephalopathy in 2013, variable phenotypes have been reported [1][2][3]. Epilepsy itself varied from early onset epileptic encephalopathy including Ohtahara syndrome, and generalized and focal epilepsies of different ages [1,4,5]. Movement phenotypes were also reported since the rst clinical report and have recently become major presenting symptoms: so far, chorea, dystonia, orofacial dyskinesia, and stereotyped hand movements have been reported in patients of different ages, associated with GNAO1 [1,[6][7][8]. Developmental milestones are also delayed in most patients with GNAO1 variants and the degree of developmental delay varies from neonatal hypotonia to intellectual disability [7][8][9]. Interestingly, most patients mainly present phenotypes between the epilepsy or movement disorder, whereas a small number of patients showed both epilepsy and movement phenotypes equally [10]. Many studies suggested that different locations or different functional changes of variants lead to separate phenotypes [1,[10][11][12]. Gain-offunction mutation (GOM) turned out to be associated with movement disorder [11]. However, it could not explain all the cases. Further studies on phenotype, genotype, and molecular pathways are certainly required for better understanding. Phenotypes often mimic other neurodevelopmental disorders and are barely immediately recognizable because the disease usually occurs in infancy with nonspeci c symptoms and evolves over time. Therefore, comprehensive and serial phenotyping would be the very rst step to grasp the disease and establish further functional studies.
Here, we report 6 pediatric patients who carried GNAO1 variants identi ed from the Korean Undiagnosed Disease Program (KUDP), with the aim of delineating detailed phenotypes and characterizing their phenotype-genotype association with a literature review.

Patients And Methods
Six patients who carried GNAO1 variants were enrolled in this study. All patients were diagnosed using whole exome sequencing (WES) after participating in the KUDP, which launched in 2017 [13]. The entire KUDP protocol including diagnostic process and data sharing was approved by the Institutional Review Board (IRB) of Seoul National University Hospital (IRB No. 1904-054-1027 and written consent forms were obtained from all parents or their legal representatives. All medical records were reviewed and home videos of patients with movement symptom were collected and analyzed independently by 2 pediatric neurologists.

Whole Exome Sequencing and Variant Identi cation
Of the 6 families, 3 underwent trio-WES and the other 3 had only the probands sequenced. Genomic DNA was extracted from peripheral blood leucocytes using a QIAamp DNA Blood Midi Kit according to the manufacturer's instructions (Qiagen, Valencia, CA, USA). WES procedures including exome capturing and sequencing were performed at Theragen Etex Bio Institute (Suwon, Korea). The sequenced reads were aligned to human reference genome patch 13 (GRCh37.p13) using a Burrows-Wheeler Aligner (version 0.7.15). Picard software (version 2.8.0), SAMtools (version 1.8), and a Genome Analysis Toolkit (GATK, version 4.1.4) were used for further data processing such as removal of polymerase chain reaction (PCR) duplicates, base recalibration, and variant quality control. All variants were called using the GATK HaplotypeCaller in GVCF mode, and the called variants were annotated using ANNOVAR and SnpEff. The pathogenicity of variants was evaluated according to the American College of Medical Genetics (ACMG) standards guidelines [14]. Segregation test was done for 3 proband-WES cases using Sanger sequencing.

Amplicon Sequencing
Amplicon sequencing was conducted for 1 family (patient 5) to identify parental mosaicism (Fig. 1). The primer was designed to amplify genomic region of interest. It can create a single amplicon of approximate 200 bp and the target position has a distance of 100 bp or less from the 5′ end. Six nucleotide barcode and adaptor sequences were added to the 5′ end of the primers to identify family members. PCR procedures were performed as previously described in detail [15]. Next-generation sequencing was performed using a dual indexing strategy and PCR free kit by Theragen Etex Bio Institute (Suwon, Korea).

Overall Clinical Features
Three female and 3 male patients were enrolled and evaluated. All clinical features are summarized in Table 1. Five patients were referred to the clinic due to hypotonia or global developmental delay which was noticed at different ages (20 months old on average, range 0-75 months). Four patients (patients 2-5) had severely retarded development and 2 of them (patients 2 and 3) could not walk independently until the last follow-up (at 7.2 and 3.3 years old, respectively). Patient 4 showed weak crying and respiratory di culty after birth, and was treated in a neonatal intensive care unit. She started unsteady gait without support at 4 years old, but no further achievement was shown till the most recent follow-up at her age of 8.8 years. Movement disorder noted in 5 patients appeared to be heterogeneous in symptoms and onset ages. Only 1 case (patient 4) had focal epilepsy, which started at 6 years old and was well controlled with valproate monotherapy. Patient 3 had neither movement disorder nor epilepsy. He presented as having infantile hypotonia and profound developmental delay. Generalized spasticity developed and progressed over time, dominantly on lower extremities. He was 3.3 years old at last examination and he could sit up slowly from a prone position. focal dystonic gait was recognized at the age of 10 years. She underwent several genetic tests for dystonia including Segawa disease, but no sequence variants in known genes were noticed. Her symptoms had progressed slowly and spasticity of lower extremities and increased knee jerk was noted at the latest clinical follow-up (17 years old

Mutation Analysis and Genotype-Phenotype Association
Six different variants from 6 patients were identi ed using WES (Table 1). Patients 1-3 underwent trio-WES whereas patients 4-6 had WES for proband only. Three variants were reported in previous articles whereas others were not and all variants were classi ed as pathogenic or likely pathogenic according to the ACMG guidelines. Five variants were con rmed to be de novo mutations, but patient 5 inherited her variant from her mother who carried the mosaic variant, a state identi ed through amplicon sequencing (Fig. 1). In this cohort, 4 variants (from patients 2, 4, 5, and 6) were located in a mutational hot spot and 3 of them (p.Glu246Lys of patient 2, p.Ala227Val of patient 4, p.Arg209His of patient 5) were reported previously [2,4,6,7]. We reviewed previously reported cases and compared detailed phenotypes (Table 2). Patients with the variant of p.Glu246Lys or p.Arg209His showed quite homogeneous phenotypes: e.g., infantile hypotonia, profound development delay, or severe choreoathetosis started in early childhood. In particular, patients with p.Arg209His were reported to have severe exacerbation and required multiple admissions. * They were dizygotic twins from non-consanguineous parents † They were siblings from non-consanguineous parents ‡ They were siblings from on-consanguineous pare

Discussion
Movement disorder is di cult to evaluate, especially in children, because they are in a developmental process and their symptoms evolve over time. Comprehensive phenotyping is quite important for diagnosis of these patients, even in the genomic era. In our cohort, all except 1 patient had multiple genetic testing including diagnostic exome sequencing and gene panel sequencing before participation in KUDP. After the diagnosis, we reviewed the patient's clinical course including previous videos. We found some quite unique presentations of patients with some recurrent GNAO1 variants (p.Glu246Lys and p.Arg209His). As previously reported, patients initially presented with profound infantile hypotonia and developmental delay, and severe choreoathetosis movement developed in all patients during early childhood [2,4,6,7]. Patient 2 in our cohort showed movement symptoms since he was about 2 years old. The symptoms persisted, but were not much progressed or suddenly exacerbated, as reported in patients with a p.Glu246Lys variant. Patient 5 was reported to have orofacial dyskinesia starting at 4 years old, but was eventually found to have early hyperkinetic movement. She was also admitted several times for pneumonia and accompanying aggravation of neck dystonia. Patients with the variant p.Arg209His also experienced several dystonic exacerbations and sometimes needed treatment in an intensive care unit [2,6]. This indicated a quite homogeneous clinical course. Most of those patients visited a clinic for their hypotonia or developmental delay, which are nonspeci c and common for a pediatric neurologist. However, if we noticed unexplained early onset dystonia or chorea as well as severe developmental delay, GNAO1 might be initially considered as a genetic cause. Early diagnosis based on clinical presentation is important because of additional treatment options. Deep brain stimulation and tetrabenazine were reported to be effective, especially for acute exacerbation [2,9,16]. Patients in this cohort have not yet been treated using those options, but these options are still considered in cases with exacerbation or rapid progression.
There were also atypical cases in our cohort. Patient 1 did not show obvious movement presentation till 10 years old. Patient 3 with the novel variant p.Aal301del showed severe developmental delay and progressive spasticity without any obvious paroxysmal movements. Patient 4, who carried the variant p.Ala227Val, presented with infantile hypotonia without early-onset epilepsy, unlike a previous report of infantile spasm and early-onset epileptic encephalopathy as a main presentation in patients with the same variant [7,17]. It is obvious that a genotype-phenotype correlation exists, at least in certain loci. Many functional studies were performed, especially for recurrent mutations to date. Some variants work as GOM, whereas others work as loss-of-function mutations [1,2,11]. Different functional alteration may contribute to different phenotypes of GNAO1 encephalopathy [11,12].
Further studies are expected to evaluate other variants in GNAO1 and its related pathway, which will allow us to know more about the disease and its possible treatment.
We veri ed maternal mosaicism from 1 family (patient 5) by additional amplicon sequencing. We suspected mosaicism in this patient, based on con rmative Sanger sequencing for the patient's mother, indicating a low heterozygous peak. This veri cation is crucial for the family counselling. Three familial cases were already reported in spite of a small number of total patients, which suggested parental mosaicism might be common in GNAO1 encephalopathy [2,4,6]. Therefore, testing for the mosaicism should be conducted if parents have plan to have another child.
In conclusion, we reported 6 cases of GNAO1 encephalopathy focusing on their movement phenotypes with their video clips (Additional les). Early-onset chorea with profound developmental problems is quite characteristic for patients with a movementdominant phenotype. We also reported atypical and novel ndings for GNAO1 encephalopathy including proven mosaicism in parents, which is important to guide genetic counselling.

Declarations
Ethics approval and consent to participate