In vitro study of ATP1A3 p.Ala275Pro mutant causing alternating hemiplegia of childhood and rapid-onset dystonia-parkinsonism.

Introduction: We previously reported that ATP1A3 c.823G>C (p.Ala275Pro) mutant causes varying phenotypes of alternative hemiplegia of childhood and rapid-onset dystonia-parkinsonism in the same family. This study aims to investigate the function of ATP1A3 c.823G>C (p.Ala275Pro) mutant at the cellular and zebrafish models. Methods: ATP1A3 wild-type and mutant Hela cell lines were constructed, and ATP1A3 mRNA expression, ATP1A3 protein expression and localization, and Na+-K+-ATPase activity in each group of cells were detected. Additionally, we also constructed zebrafish models with ATP1A3 wild-type overexpression (WT) and p.Ala275Pro mutant overexpression (MUT). Subsequently, we detected the mRNA expression of dopamine signaling pathway-associated genes, Parkinson's disease-associated genes, and apoptosisassociated genes in each group of zebrafish, and observed the growth, development, and movement behavior of zebrafish. Results: Cells carrying the p.Ala275Pro mutation exhibited lower levels of ATP1A3 mRNA, reduced ATP1A3 protein expression, and decreased Na+-K+-ATPase activity compared to wild-type cells. Immunofluorescence analysis revealed that ATP1A3 was primarily localized in the cytoplasm, but there was no significant difference in ATP1A3 protein localization before and after the mutation. In the zebrafish model, both WT and MUT groups showed lower brain and body length, dopamine neuron fluorescence intensity, escape ability, swimming distance, and average swimming speed compared to the control group. Moreover, overexpression of both wild-type and mutant ATP1A3 led to abnormal mRNA expression of genes associated with the dopamine signaling pathway and Parkinson's disease in zebrafish, and significantly upregulated transcription levels of bad and caspase-3 in the apoptosis signaling pathway, while reducing the transcriptional level of bcl-2 and the bcl-2/bax ratio. Conclusion: This study reveals that the p.Ala275Pro mutant decreases ATP1A3 protein expression and Na+/K+-ATPase activity. Abnormal expression of either wild-type or mutant ATP1A3 genes impairs growth, development, and movement behavior in zebrafish.

Epidemiological study on pediatric-onset dystonia in Japan: A questionnaire-based survey.

OBJECTIVE: This study aimed to investigate the clinical characteristics of pediatric-onset dystonia in Japan, addressing the diagnostic challenges arising from symptom variations and etiological diversity. METHODS: From 2020 to 2022, questionnaires were distributed to 1218 board certified child neurologists (BCCNs) by Japanese Society of Child Neurology. In the primary survey, participants were asked to report the number of patients with pediatric-onset dystonia under their care. Subsequently, the follow-up secondary survey sought additional information on the clinical characteristics of these patients. RESULTS: The primary survey obtained 550 responses (response rate: 45 %) from BCCNs for their 736 patients with dystonia. The predominant etiologies included inherited cases (with DYT10   being the most prevalent, followed by DYT5 and ATP1A3-related neurologic disorders), acquired cases (with perinatal abnormalities being the most common), and idiopathic cases. The secondary survey provided clinical insights into 308 cases from 82 BCCNs. Infancy-onset dystonia presented as persistent and generalized with diverse symptoms, primarily linked to ATP1A3-related neurologic disorders and other genetic disorders resembling acquired dystonia. Conversely, childhood/adolescent-onset dystonia showed paroxysmal, fluctuating courses, predominantly affecting limbs. The most common etiologies were DYT5 and DYT10 , leading to therapeutic diagnoses. CONCLUSION: Pediatric-onset dystonia in Japan was treated by 28 % of BCCNs. The majority of cases were inherited, with high prevalence rates of DYT5 and DYT10 . Infancy-onset dystonia exhibits diverse etiologies and symptoms, emphasizing the utility of various examinations, including genetic testing. These findings significantly contribute to our understanding of pediatric-onset dystonia in Japan, although this study has the limitation of questionnaire survey.

ATP1A3 regulates protein synthesis for mitochondrial stability under heat stress.

Pathogenic variants in ATP1A3, the gene encoding the α3 subunit of the Na+/K+-ATPase, cause alternating hemiplegia of childhood (AHC) and related disorders. Impairments in Na+/K+-ATPase activity are associated with the clinical phenotype. However, it remains unclear whether additional mechanisms are involved in the exaggerated symptoms under stressed conditions in patients with AHC. We herein report that the intracellular loop (ICL) of ATP1A3 interacted with RNA-binding proteins, such as Eif4g (encoded by Eif4g1), Pabpc1 and Fmrp (encoded by Fmr1), in mouse Neuro2a cells. Both the siRNA-mediated depletion of Atp1a3 and ectopic expression of the p.R756C variant of human ATP1A3-ICL in Neuro2a cells resulted in excessive phosphorylation of ribosomal protein S6 (encoded by Rps6) and increased susceptibility to heat stress. In agreement with these findings, induced pluripotent stem cells (iPSCs) from a patient with the p.R756C variant were more vulnerable to heat stress than control iPSCs. Neurons established from the patient-derived iPSCs showed lower calcium influxes in responses to stimulation with ATP than those in control iPSCs. These data indicate that inefficient protein synthesis contributes to the progressive and deteriorating phenotypes in patients with the p.R756C variant among a variety of ATP1A3-related disorders.

Childhood fever and hearing loss associated with CAPOS syndrome.

CAPOS (cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss) syndrome is a rare genetic disorder caused by the heterozygous mutation, c.2452G > A, in the ATP1A3 gene. CAPOS syndrome involves a characteristic episode in which neuropathy develops after a fever in childhood, and here, we describe the case of a patient with CAPOS syndrome. The patient had repeated episodes of a fever around 74 months of age. Although he could speak at 23 months of age, he presented with hearing difficulty after the fever. Pure-tone audiometry revealed moderate-to-severe bilateral sensorineural hearing loss, and auditory brainstem response (ABR) showed poor response in the both ears. Auditory stead-state response (ASSR) produced relatively consistent results compared to pure-tone audiometry. A mutation in the ATP1A3 gene was detected through genetic testing. In CAPOS syndrome, a genetic mutation leads to desynchronization during neural firing. We believe that this desynchronization in neural firing is responsible for the lack of response in the ABR and the presence of a response in the ASSR. In this patient, we attribute the response detection in ASSR to its greater tolerance for errors in the timing of neural firing compared to ABR.

Analysis of the sodium pump subunit ATP1A3 in glioma patients: Potential value in prognostic prediction and immunotherapy.

The ATP1A3 gene is associated with the development and progression of neurological diseases. However, the pathological function and therapeutic value of ATP1A3 in glioblastoma (GBM) remains unknown. In this study, we tried to explore the correlation between the ATP1A3 gene expression and immune features in GBM samples. We found that ATP1A3 gene expression levels showed significant negative correlation with immune checkpoints such as PD-L1, CTLA-4 and IDO1. Next, ATP1A3 gene expression levels showed significant negative correlation with the anti-cancer immune cell process, the immune score and stromal score. By grouping ATP1A3 expression levels, we found that that immunomodulator-related genes and tumor-associated immune cell effector gene expression levels were associated with lower ATP1A3 expression. In addition, immunotherapy prediction pathway activity and a majority of the anti-cancer immune cell process activity levels were also showed to be correlated with lower ATP1A3 gene expression. Further, nine prognostic factors were identified by prognostic analysis, and a GBM prognostic model (risk score) was established. We applied the model to the TCGA GBM training set sample and the GSE4412 validation set sample and found that patients in the high risk score subgroup had significantly shorter survival time, demonstrating the prognostic value and prognostic efficacy of the risk score. Furthermore, ATP1A3 overexpression has also been found to sensitize cancer cells to anti-PD-1 therapy. In conclusion, we showed that ATP1A3 is a highly promising treatment target in GBM and the risk score is an independent prognostic factor for cancer and can be used to help guide the prediction of survival time in patients with GBM.

[Clinical features of CAPOS syndrome caused by maternal ATP1A3 gene variation: a case report].

CAPOS syndrome is an autosomal dominant neurological disorder caused by mutations in the ATP1A3 gene. Initial symptoms, often fever-induced, include recurrent acute ataxic encephalopathy in childhood, featuring cerebellar ataxia, optic atrophy, areflflexia, sensorineural hearing loss, and in some cases, pes cavus. This report details a case of CAPOS syndrome resulting from a maternal ATP1A3 gene mutation. Both the child and her mother exhibited symptoms post-febrile induction,including severe sensorineural hearing loss in both ears, ataxia, areflexia, and decreased vision. Additionally, the patient's mother presented with pes cavus. Genetic testing revealed a c. 2452G>A（Glu818Lys） heterozygous mutation in theATP1A3 gene in the patient . This article aims to enhance clinicians' understanding of CAPOS syndrome, emphasizing the case's clinical characteristics, diagnostic process, treatment, and its correlation with genotypeic findings.

Childhood-related neural genotype-phenotype in ATP1A3 mutations: comprehensive analysis.

BACKGROUND: ATP1A3 is a gene that encodes the ATPase Na + /K + transporting subunit alpha-3 isoenzyme that is widely expressed in GABAergic neurons. It maintains metabolic balance and neurotransmitter movement. These pathways are essential for the proper functioning of the nervous system. A mutation in the ATP1A3 gene demonstrates remarkable genotype-phenotype heterogeneity. OBJECTIVES: To provide insight into patients with ATP1A3 mutation. MATERIAL AND METHODS: These cases were identified using next generation sequencing. The patients' clinical and genetic data were retrieved. Detailed revision of the literature was conducted to illustrate and compare findings. The clinical, genetical, neuroimaging, and electrophysiological data of all pediatric patients were extracted. RESULTS: The study included 14 females and 12 males in addition to two novel females cases. Their mean current age is 6.3 ± 4.24 years. There were 11.54% preterm pregnancies with 5 cases reporting pregnancy complications. Mean age of seizure onset was 1.07 ± 1.06 years. Seizure semiology included generalized tonic-clonic, staring spells, tonic-clonic, and others. Levetiracetam was the most frequently used Anti-seizure medication. The three most frequently reported classical symptoms included alternating hemiplegia of childhood (50%), cerebellar ataxia (50%), and optic atrophy (23.08%). Non-classical symptoms included dystonia (73.08%), paroxysmal dyskinesias (34.62%), and encephalopathy (26.92%). Developmental delay was reported among 84.62% in cognitive, 92.31% in sensorimotor, 80.77% in speech, and 76.92% in socioemotional. EEG and MRI were non-specific. CONCLUSION: Our study demonstrated high heterogeneity among patients with pathogenic variants in the ATP1A3 gene. Such variation is multifactorial and can be a predisposition of wide genetic and clinical variables. Many patients shared few similarities in their genetic map including repeatedly reported de novo, heterozygous, mutations in the gene. Clinically, higher females prevalence of atypical presentation was noted. These findings are validated with prior evidence and the comprehensive analysis in this study.

Cilostazol treats transient heart failure caused by ATP1A3 variant-associated polymicrogyria.

BACKGROUND: Some patients with ATP1A3 variant-associated polymicrogyria have recurrent transient heart failure. However, effective treatment for the transient cardiac condition remains to be elucidated. CASE REPORT: The patient started experiencing focal motor onset seizures in 12 h after birth, revealing bilateral diffuse polymicrogyria. The patient also experienced transient bradycardia (sinus bradycardia) attacks from 15 days old. Echocardiography revealed a reduced ejection fraction; however, no obvious electrocorticogram or electroencephalogram abnormalities were observed during the attacks. Initially, the attacks occurred in clusters daily. They later decreased in frequency, occurring at monthly intervals. Repeated episodes of transient bradycardia attacks and polymicrogyria indicated possible ATP1A3 gene abnormality and genetic testing revealed a novel heterozygous ATP1A3 variant (NM_152296: exon22:c.2977_2982del:p.(Glu993_Ile994del)), which was not found in the patient's parents. Cilostazol was administered at 3 months old for recurrent transient bradycardia attacks. Cilostazol significantly shortened the duration of bradycardia episodes and prolonged the interval between attacks. Cilostazol also effectively treats transient symptomatic bradycardia. CONCLUSION: Cilostazol could be a treatment option for recurrent transient bradycardia attacks associated with ATP1A3 gene abnormalities and polymicrogyria.

Characteristics of non-sleep related apneas in children with alternating hemiplegia of childhood.

BACKGROUND: Non-sleep related apnea (NSA) has been observed in alternating hemiplegia of childhood (AHC) but has yet to be characterized. GOALS: Investigate the following hypotheses: 1) AHC patients manifest NSA that is often severe. 2) NSA is usually triggered by precipitating events. 3) NSA is more likely in patients with ATP1A3 mutations. METHODS: Retrospective review of 51 consecutive AHC patients (ages 2-45 years) enrolled in our AHC registry. NSAs were classified as mild (not needing intervention), moderate (needing intervention but not perceived as life threatening), or severe (needing intervention and perceived as life threatening). RESULTS: 19/51 patients (37 %) had 52 NSA events (6 mild, 11 moderate, 35 severe). Mean age of onset of NSA (± Standard Error of the Mean (SEM)): 3.8 ± 1.5 (range 0-24) years, frequency during follow up was higher at younger ages as compared to adulthood (year 1: 2.2/year, adulthood: 0.060/year). NSAs were associated with triggering factors, bradycardia and with younger age (p < 0.008 in all) but not with mutation status (p = 0.360). Triggers, observed in 17 patients, most commonly included epileptic seizures in 9 (47 %), anesthesia, AHC spells and intercurrent, stressful, conditions. Management included use of pulse oximeter at home in nine patients, home oxygen in seven, intubation/ventilatory support in seven, and basic CPR in six. An additional patient required tracheostomy. There were no deaths or permanent sequalae. CONCLUSIONS: AHC patients experience NSAs that are often severe. These events are usually triggered by seizures or other stressful events and can be successfully managed with interventions tailored to the severity of the NSA.

Alternating hemiplegia of childhood.

Alternating hemiplegia of childhood (AHC) is characterized by recurrent episodes of hemiplegia which may alternate sides between attacks. The condition is associated with severe neurodevelopmental disorder presenting in early infancy, and may encompass a wide range of other paroxysmal manifestations (e.g., dystonia, nystagmus, dysautonomia) and pervasive neurological disabilities (e.g., developmental delay, learning disabilities, choreoathetosis, and ataxia). Epileptic seizures are particularly common among patients with AHC. Diagnosis is usually based on history and clinical grounds using the Aicardi criteria. Mutations in the ATP1A3 gene are implicated in the disease pathology of the condition, as well as several other neurodevelopmental disorders, suggesting AHC forms part of a spectrum of overlapping clinical syndromes rather than a distinct clinical entity per se. Management of patients with AHC includes the rapid induction of sleep during paroxysmal attacks and the avoidance of identified triggers. Pharmacotherapeutic treatments have a role in managing epileptic seizures, as well as in the prevention of paroxysmal attacks wherein flunarizine remains the treatment of choice.

ATP1A3 related disease manifesting as rapid onset dystonia-parkinsonism with prominent myoclonus and exaggerated startle.

We report ATP1A3-associated rapid-onset dystonia-parkinsonism with an atypical presentation including myoclonus and exaggerated startle in four patients. Their prominence over parkinsonism prompted consideration of a syndromic diagnosis of myoclonus dystonia. ATP1α3 dysfunction in GABAergic neurons could explain these examination findings. The spectrum of ATP1A3-associated movement disorders includes myoclonus-dystonia.

Brain structural changes in alternating hemiplegia of childhood using single-case voxel-based morphometry analysis.

BACKGROUND AND PURPOSE: Alternating hemiplegia of childhood (AHC) is a rare neurodevelopmental disease caused by ATP1A3 mutations. Using voxel-based morphometry (VBM) analysis, we compared an AHC patient cohort with controls. Additionally, with single-case VBM analysis, we assessed the associations between clinical severity and brain volume in patients with AHC. MATERIALS AND METHODS: To investigate structural brain changes in gray matter (GM) and white matter (WM) volumes between 9 patients with AHC and 20 age-matched controls, VBM analysis was performed using three-dimensional T1-weighted magnetic resonance imaging. Single-case VBM analysis was also performed on nine patients with AHC to investigate the associations between the respective volumes of GM/WM differences and the motor level, cognitive level, and status epilepticus severity in patients with AHC. RESULTS: Compared with controls, patients with AHC showed significant GM volume reductions in both hippocampi and diffuse cerebellum, and there were WM reductions in both cerebral hemispheres. In patients with AHC, cases with more motor dysfunction, the less GM/WM volume of cerebellum was shown. Three of the six cases with cognitive dysfunction showed a clear GM volume reduction in the insulae. Five of the six cases with status epilepticus showed the GM volume reduction in hippocampi. One case had severe status epilepticus without motor dysfunction and showed no cerebellar atrophy. CONCLUSION: With single-case VBM analysis, we could show the association between region-specific changes in brain volume and the severity of various clinical symptoms even in a small sample of subjects.

Precision therapy for a medically actionable ATP1A3 variant from a genomic medicine program in an underserved population.

BACKGROUND: Genomic medicine is revolutionizing the diagnosis of rare diseases, but the implementation has not benefited underrepresented populations to the same degree. Here, we report the case of a 7-year-old boy with hypotonia, global developmental delay, strabismus, seizures, and previously suspected mitochondrial myopathy. This proband comes from an underrepresented minority and was denied exome sequencing by his public insurance. METHODS: After informed consent was obtained, buccal cells from the proband were collected and whole exome sequencing was performed. Illumina Dragen and Emedgene software was used to analyze the data at Baylor Genetics. The variants were further intepreted according to ACMG guidelines and the patient's phenotype. RESULTS: Through whole-exome sequencing (WES) under the Community Texome project, he was found to have a heterozygous de novo pathogenic variant in the ATP1A3 gene located on chromosome 19q13. CONCLUSION: In retrospect, his symptomatology matches the known medical conditions associated with the ATP1A3 gene namely Alternating Hemiplegia of Childhood 2 (AHC), a rare autosomal dominant disorder with an incidence of 1 in one million. His single nucleotide variant, (c.2401G>A, p.D801N), is predicted to be damaging. The specific amino acid change p.D801N has been previously reported in ClinVar along with the allelic variant p.D801Y and both are considered pathogenic. The identification of this variant altered medical management for this patient as he was started on a calcium antagonist and has reported no further hemiplegic episodes. This case illustrates the value of implementing genomic medicine for precision therapy in underserved populations.

Epilepsy with eyelid myoclonia in the setting of de novo pathogenic variant in ATP1A3.

Mutations in the ATP1A3 gene have been associated with several syndromes, including rapid-onset dystonia-parkinsonism, alternating hemiplegia of childhood, and cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss. In this clinical commentary, we report a 2-year-old female patient with de novo pathogenic variant in the ATP1A3 gene associated with an early-onset form of epilepsy with eyelid myoclonia. The patient had frequent eyelid myoclonia occurring 20-30 times per day, without loss of awareness or other motor manifestations. EEG showed generalized polyspikes and spike-and-wave complexes maximal in the bifrontal regions, with prominent eye closure sensitivity. A sequencing-based epilepsy gene panel revealed a de novo pathogenic heterozygous variant in ATP1A3. The patient showed some response to flunarizine and clonazepam. This case highlights the importance of considering ATP1A3 mutations in the differential diagnosis of early-onset epilepsy with eyelid myoclonia and the potential benefit of flunarizine in improving language and coordination development in patients with ATP1A3-related disorders.

Evaluating Dysfunction in Fever-Induced Paroxysmal Weakness and Encephalopathy.

Heterozygous variants in the ATP1A3 gene are linked to well-known neurological phenotypes. There has been growing evidence for a separate phenotype associated with variants in residue Arg756-fever-induced paroxysmal weakness and encephalopathy (FIPWE) or relapsing encephalopathy with cerebellar ataxia (RECA). With only about 20 cases being reported, the clinical features associated with mutations at Arg756 have not been fully elucidated. We report a case of FIPWE with a p.Arg756Cys change in the ATP1A3 gene and a comparison of the clinical features, including electrophysiological examination, with previous cases. The 3-year-old male patient had normal psychomotor development, presenting with recurrent symptoms of generalized hypotonia with loss of gait, mutism, and dystonic movements only during febrile illnesses since 19 months of age. At 2.7 years of age, a third neurological decompensation episode occurred, during which electroencephalography (EEG) did not reveal high voltage slow waves or epileptiform discharge. Nerve conduction studies (NCS) also did not show latency delay or amplitude reduction. ATP1A3 exon sequencing showed a heterozygous p.Arg756Cys mutation. While the patient experienced repeated encephalopathy-like episodes, including severe hypotonia during febrile illness, EEG and NCS did not reveal any obvious abnormalities. These electrophysiological findings may represent an opportunity to suspect FIPWE and RECA.

Cation leak through the ATP1A3 pump causes spasticity and intellectual disability.

ATP1A3 encodes the α3 subunit of the sodium-potassium ATPase, one of two isoforms responsible for powering electrochemical gradients in neurons. Heterozygous pathogenic ATP1A3 variants produce several distinct neurological syndromes, yet the molecular basis for phenotypic variability is unclear. We report a novel recurrent variant, ATP1A3(NM_152296.5):c.2324C>T; p.(Pro775Leu), in nine individuals associated with the primary clinical features of progressive or non-progressive spasticity and developmental delay/intellectual disability. No patients fulfil diagnostic criteria for ATP1A3-associated syndromes, including alternating hemiplegia of childhood, rapid-onset dystonia-parkinsonism or cerebellar ataxia-areflexia-pes cavus-optic atrophy-sensorineural hearing loss (CAPOS), and none were suspected of having an ATP1A3-related disorder. Uniquely among known ATP1A3 variants, P775L causes leakage of sodium ions and protons into the cell, associated with impaired sodium binding/occlusion kinetics favouring states with fewer bound ions. These phenotypic and electrophysiologic studies demonstrate that ATP1A3:c.2324C>T; p.(Pro775Leu) results in mild ATP1A3-related phenotypes resembling complex hereditary spastic paraplegia or idiopathic spastic cerebral palsy. Cation leak provides a molecular explanation for this genotype-phenotype correlation, adding another mechanism to further explain phenotypic variability and highlighting the importance of biophysical properties beyond ion transport rate in ion transport diseases.

The role of ATP1A3 gene in epilepsy: We need to know more.

The ATP1A3 gene, which encodes the Na+/K+-ATPase α3 catalytic subunit, plays a crucial role in both physiological and pathological conditions in the brain, and mutations in this gene have been associated with a wide variety of neurological diseases by impacting the whole infant development stages. Cumulative clinical evidence suggests that some severe epileptic syndromes have been linked to mutations in ATP1A3, among which inactivating mutation of ATP1A3 has been intriguingly found to be a candidate pathogenesis for complex partial and generalized seizures, proposing ATP1A3 regulators as putative targets for the rational design of antiepileptic therapies. In this review, we introduced the physiological function of ATP1A3 and summarized the findings about ATP1A3 in epileptic conditions from both clinical and laboratory aspects at first. Then, some possible mechanisms of how ATP1A3 mutations result in epilepsy are provided. We think this review timely introduces the potential contribution of ATP1A3 mutations in both the genesis and progression of epilepsy. Taken that both the detailed mechanisms and therapeutic significance of ATP1A3 for epilepsy are not yet fully illustrated, we think that both in-depth mechanisms investigations and systematic intervention experiments targeting ATP1A3 are needed, and by doing so, perhaps a new light can be shed on treating ATP1A3-associated epilepsy.