To underline the pathogenicity of five novel heterozygous missense variants in ATP1A1 using a specific bioassay and to characterize a complex neurodevelopmental syndrome, broadening the known phenotypic spectrum associated with this gene.

ATP1A1 encodes the alpha subunit of the sodium-potassium ATPase. Pathogenic variants in ATP1A1 have been associated with an autosomal dominant Charcot-Marie-Tooth (CMT) phenotype as well as with spastic paraplegia, hypomagnesemia, mental retardation, and seizures (OMIM). According to the GnomAD constraint metrics, ATP1A1 has an unusually low tolerance to missense (Z = 6.22) and loss of function (pLI = 1) mutations.

Patients were examined by trained neurologists, and written informed consent was obtained following local legal guidelines. Variants were identified by whole exome or whole genome sequencing, and co-segregation analyses were conducted using Sanger sequencing. Variant pathogenicity was assessed via ouabain survival assay. HEK cells were transfected with ouabain-resistant plasmids encoding the five ATP1A1 variants. Cells were treated with the ATPase inhibitor ouabain for 72h at a concentration of 0.5µM, and cell survival was measured by luciferase assay.

In five unrelated children, we identified five novel heterozygous de novo variants in ATP1A1 (ENST00000295598.10; ): ;p.Gln225Arg, ;p.Gly335Cys, ;p.Gly509Asp, c.2152G>A;p.Gly718Ser, and ;p.Phe923Tyr that were all not present in healthy control populations (GnomAD). Patients had a complex phenotype comprising intellectual disability, spasticity, and motor predominant peripheral neuropathy, and age at onset in early childhood. Additional features included sensory loss, sleep disturbances, and seizures. In HEK cells transfected with ouabain-insensitive ATP1A1 constructs, cell viability was significantly decreased in mutants treated with the ATPase inhibitor ouabain.

Replicating the haploinsufficiency mechanism of disease with a gene-specific assay, we confirmed the pathogenicity of the five novel variants. We conclude that the phenotypic spectrum associated with pathogenic ATP1A1 mutations is broader than previously described.