UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

Objective: The objective of this study is to analyze the genotype-phenotype correlation of patients with auditory neuropathy (AN), which is a clinical condition featuring normal cochlear responses and abnormal neural responses, and ATP1A3 c.2452 G > A (p.E818K), which has been generally recognized as a genetic cause of cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome. Methods: Four patients diagnosed as AN by clinical evaluation and otoacoustic emission and auditory brainstem responses were recruited and analyzed by next-generation sequencing to identify candidate disease-causing variants. Sanger sequencing was performed on the patients and their parents to verify the results, and short tandem repeat-based testing was conducted to confirm the biological relationship between the parents and the patients. Furthermore, cochlear implantation (CI) was performed in one AN patient to reconstruct hearing. Results: Four subjects with AN were identified to share a de novo variant, p.E818K in the ATP1A3 gene. Except for the AN phenotype, patients 1 and 2 exhibited varying degrees of neurological symptoms, implying that they can be diagnosed as CAPOS syndrome. During the 15 years follow-up of patient 1, we observed delayed neurological events and progressive bilateral sensorineural hearing loss in pure tone threshold (pure tone audiometry, PTA). Patient 2 underwent CI on his left ear, and the result was poor. The other two patients (patient 3 and patient 4, who were 8 and 6 years old, respectively) denied any neurological symptoms. Conclusion: ATP1A3 p.E818K has rarely been documented in the Chinese AN population. Our study confirms that p.E818K in the ATP1A3 gene is a multiethnic cause of AN in Chinese individuals. Our study further demonstrates the significance of genetic testing for this specific mutation for identifying the special subtype of AN with somewhat favorable CI outcome and offers a more accurate genetic counseling about the specific de novo mutation.