Novel mutations in ATP1A3 associated with catastrophic early life epilepsy, episodic prolonged apnea, and postnatal microcephaly

Abstract

Objective: Mutations of ATP1A3 have been associated with rapid onset dystonia-parkinsonism and more recently with alternating hemiplegia of childhood. Here we report one child with catastrophic early life epilepsy and shortened survival, and another with epilepsy, episodic prolonged apnea, postnatal microcephaly, and severe developmental disability. Novel heterozygous mutations (p.Gly358Val and p.Ile363Asn) were identified in ATP1A3 in these children.

Methods: Subjects underwent next-generation sequencing under a research protocol. Clinical data were collected retrospectively. The biochemical effects of the mutations on ATP1A3 protein function were investigated. Postmortem neuropathologic specimens from control and affected subjects were studied.

Results: The mutations localized to the P domain of the Na,K-ATPase α3 protein, and resulted in significant reduction of Na,K-ATPase activity in vitro. We demonstrate in both control human brain tissue and that from the subject with the p.Gly358Val mutation that ATP1A3 immunofluorescence is prominently associated with interneurons in the cortex, which may provide some insight into the pathogenesis of the disease.

Significance: The findings indicate these mutations cause severe phenotypes of ATP1A3-related disorder spectrum that include catastrophic early life epilepsy, episodic apnea, and postnatal microcephaly.

Keywords: ATP1A3; Apnea; Early life epilepsy; K-ATPase; Na; Next-generation sequencing; Postnatal microcephaly.

Figure 1

Representative EEG tracing from subject LR11-328 at 2 months showing left hemispheric electrographic seizure (A). Head size measurements showed postnatal onset microcephaly (B). Brain MRI of subject LR11-328 showing progressive cortical and subcortical atrophy at 5 months on sagittal (C) and axial views (D), compared to sagittal (E) and axial views (F) at age 12 months. The corpus callosum remained thin, with cortical and anterior cerebellar vermis volume loss.

Figure 2

EEG tracing from subject LR11-147 with a right hemispheric electrographic seizure associated with eye deviation and apnea (A). Brain MRI at 1 year in subject LR11-147 (B) was unremarkable. At 3.5 years of age he was encephalopathic, with nondysmorphic facies (C,D).

Figure 3

Chromatographs of Sanger sequencing in subject LR11-328 (A) and LR11-147 (B) showing de novo status of mutations c.1073G>T and c.1088T>A in ATP1A3. The resulting amino acid substitutions Gly358Val (yellow spacefill) and Ile363Asn (blue spacefill) localize to the P domain of the ATP1A3 protein (red stick representation). A beta-strand (thin ribbon backbone highlighted white) connects the mutated residues to the active site phosphorylatable Asp366 (green spacefill). The structure is tilted to reveal the linear relationship between the mutations and the active site aspartate. The β subunit (green stick representation) is at the extracellular surface, and the yellow and gold A and N domains, like the red P domain, are cytoplasmic.

Figure 4

Deficiency of Na,K-ATPase function in HEK-293 cell transfectants. HEK-293 cells were transfected with lipofectamine alone as a procedural control (A); ouabain-resistant human ATP1A3 cDNA vector without mutation (B); or ouabain-resistant vector with Gly358Val mutation (C). Twenty-four hours after transfection, 0.5 μM ouabain was added to inhibit the endogenous ATP1A1 (α1). The images show that after an additional 3 days in culture, both the control cells and the Gly358Val cells were dead. A few dead cells were present in cells transfected with wild type-ouabain resistant vector (B), but a majority were alive and migrating out from the edge. Identical complete cell death was obtained with the Ile363Asn mutation (data not shown). Mutant protein expression (D). Lysates of cells were analyzed with α3-specific antibody. C- is lysate from control HEK-293 cells, and the faint band is presumed to be slight crossreactivity of the antibody with endogenous α1. C+ is lysate of stably-transfected cells expressing ouabain-resistant WT α3 at a level that supports cell growth in the presence of 0.5 μM ouabain selection at the same rate as the parent cell line. WT, G358V, and I363N are lysates of transient transfections 48 hours after addition of plasmid, with no ouabain present. Data are representative of 3 experiments.

Figure 5

ATP1A3 immunofluorescence was localized to GABAergic cells in human temporal cortex and basal ganglia. A,B) Control (A) and ATP1A3 Gly358Val (B) cortex. Filled arrows indicate cells with interneuron morphology and perisomatic ATP1A3 IF; open arrowheads indicate outlines of cells with pyramidal morphology and absence of ATP1A3 IF. C,D) ATP1A3 (green) co-localizes with the interneuron marker GAD 65/67 (red) in control (C) and Gly358Val (D) human cortex. E,F) ATP1A3 colocalization with GAD 65/67 in human caudate nucleus. Scale bars: 50 μM