De novo ATP1A3 variants cause polymicrogyria

Figures

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  Fig. 1. Bilateral perisylvian polymicrogyria associated with de novo ATP1A3 mutations. Brain MRI scans of patient 1 [(A) and (B) at 1 day old, (C) and (D) at 2 years and 10 months], patient 3 [(E) and (F) at 2 months old, (G) and (H) at 8 months], patient 4 [(I) and (J) at 3 days old, (K) and (L) at 4 months], patient 5 [(M) and (N) at 2 days old], patient 6 [(O) and (P) at 2 months old], patient 7 [(Q) and (R) at 5 months old, (S) and (T) at 3 years old], patient 8 [(U) and (V) at 2 months old], and a neurologically normal control [(W) and (X) at 3 years old]. All images are T2-weighted axial brain MRI, except for a T2-weighted sagittal MRI (M), a fluid-attenuated inversion recovery axial MRI (N), and an axial cranial CT (S and T). Brain MRI showed irregular small gyri with apparent cortical thickening, compatible with polymicrogyria, over the perisylvian cortex (arrowheads). Patients 1, 3, and 4 (A to L) showed progressive atrophic changes with dilation of the subarachnoid space and lateral ventricles.

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  Fig. 2. Structural considerations of the identified mutations in human Na+/K+ ATPase α-subunits. (A) The crystal structure of Na+/K+ ATPase, composed of α- (pink), β- (cyan), and γ-subunits (green), from Squalus acanthias (PDB code: 2ZXE) is shown with the residues (magenta spheres) corresponding to the identified variants in humans. The squares (b to e) correspond to the magnified areas (B to E), respectively. In the right structure, spheres indicate the residues corresponding to the previously reported variants associated with AHC (orange), RDP (cyan), both AHC and RDP (green), and other phenotypes (yellow). (B) The p.Arg182 (cyan) of the β-subunit interacting with p.Gln895 (magenta) in a loop of p.Asp882–Trp896 (red wire) of the α-subunit via hydrogen bonds (black dotted lines). The residue numbering is based on human Na+/K+ ATPase. (C) The interactions between α- and β-subunits around the p.Gly854 to Phe857 region of the α-subunit. The side chains of p.Phe856 and p.Phe857 and their interacting residues (β-subunit) are shown by sticks with translucent van der Waals spheres. The conformation of p.Met57 (β-subunit) in humans was modeled instead of leucine in the S. acanthias structure.…

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  Fig. 3. Previously reported and newly identified ATP1A3 variants in a schematic of Na+/K+ ATPase α3-subunits and variant distribution among the phenotypes. (A) The black bars represent transmembrane domains (TM1 to TM10), while the lines represent the intracellular and extracellular domains of the Na+/K+ ATPase α3-subunit. A hatchet-shaped light brown structure depicts the β-subunit. The symbols and colors used for explaining variants are described in the legend. Numbers indicate the residue number at each site. A, N, and P stand for the actuator, nucleotide binding, and phosphorylation regions, respectively. Large circles represent the commonly observed variants. (B) Graph of nontruncating variant distribution of AHC, RDP, CAPOS, others, and polymicrogyria. X axis: amino acid number; y axis: each phenotype. Dashed and dotted lines indicate the median and quartile, respectively. **P < 0.01 and ***P < 0.001 using the Kolmogorov-Smirnov test. PMG, polymicrogyria; others: phenotypes other than AHC, CAPOS, RDP, and polymicrogyria, including developmental and epileptic encephalopathy, such as EIEE with or without apnea, relapsing encephalopathy with cerebellar ataxia, or fever-induced…

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  Fig. 4. Functional consequences of the identified ATP1A3 variants. Black, blue, and purple/red bars indicate the wild-type (WT), AHC/RDP-associated, and polymicrogyria-associated variants, respectively. The upper right box shows color-coded information. Error bars, means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. (A) ATPase activity in COS-7 cells. N = 3. (B) Expression of myc-tagged ATP1A3 and HaloTag-fused ATP1B1 in human embryonic kidney (HEK) 293T cells. Left: A representative blot. Right: The relative ATP1A3 and mature β1-subunit expression, normalized to the wild type. N = 3. WB, Western blot. (C) Coimmunoprecipitation of myc-tagged ATP1A3 and HaloTag-fused ATP1B1 in HEK293T cells. Left: A representative blot. Middle: The relative ATP1B1 recovery rate. N = 3. Right: The proportion of N-glycosylated ATP1B1 bound to ATP1A3. (D) The total HEK293T cell components cotransfected with ATP1A3 (wild type, p.Ile758Ser, or p.Gln895Pro) and ATP1B1 cDNA vectors were separated into three fractions: cytosol, organelles, and plasma membrane. Left: A representative blot. A dashed rectangle denotes Western blotting on a different cartridge. The second panel from the left: The relative ATP1A3…

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  Fig. 5. Colocalization of ATP1A3 and ATP1B1 at the cis-/trans-Golgi apparatus and plasma membrane. Through (A) to (C), the left and right panels show immunofluorescence under the cotransfection of wild-type ATP1A3 and wild-type ATP1B1 plasmids and p.Gln895Pro ATP1A3 and wild-type ATP1B1 plasmids, respectively. Scale bars, 5 μm. (A) ATP1A3 and ATP1B1 colocalizing at the cis-Golgi network in both panels (arrowhead). (B) ATP1A3 and ATP1B1 colocalizing at the trans-Golgi network in both panels (arrowhead). (C) ATP1A3 and ATP1B1 colocalizing at the plasma membrane in both panels (arrowhead). For (A) and (B), lower images are the pseudocolored maps of the product of the differences from the mean analysis (PDM maps) for the respective upper images. For (C), lower right panels are the PDM maps for the upper two images. In the PDM maps, positive correlation is indicated in yellow, negative correlation in blue, and random distribution in black.

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  Fig. 6. In situ PLA fluorescence study showing decreased interaction between ATP1A3 and ATP1B1 with the polymicrogyria-associated p.Gln895Pro variant. (A) The production of in situ PLA fluorescent spots (Duolink) indicates the subcellular sites where myc-tagged ATP1B1 and HaloTag-fused ATP1B1 colocalize within 40 nm. Immunofluorescence study showing that Duolink signals were substantially decreased at HaloTag-fused ATP1B1 expressed in the plasma membrane in HEK293T cells cotransfected with p.Gln895Pro ATP1A3 and ATP1B1 plasmids, compared with those with p.Ile758Ser ATP1A3/ATP1B1 and wild-type ATP1A3/ATP1B1 plasmids. Scale bars, 20 μm. DAPI, 4′,6-diamidino-2-phenylindole. (B) Mean intensity in the specified region was significantly decreased in HEK293T cells cotransfected with p.Gln895Pro ATP1A3 and ATP1B1 plasmids, while those with p.Ile758Ser ATP1A3/ATP1B1 did not appear to differ in mean signal intensity compared with wild-type ATP1A3/ATP1B1. We confirmed this tendency with replicate experiments (N = 4). a.u., arbitrary units.

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  Fig. 7. Migration defects in ATP1A3 p.Asp992del variant–expressing neurons in the embryonic mouse cortex. (A) Schematic of the study design using mice. (B and C) Plasmids expressing the wild-type, p.Asp992del, p.Gln895Pro, and p.Ile758Ser variants of ATP1A3 were introduced, together with GFP-expressing plasmids, into the ventricles of mice on E14.5. After in utero electroporation, the embryonic brains were collected and fixed at E18.5, and migration was quantified using cell distributions. The total cortical thickness was divided into five equal parts, named BIN1 to BIN5. The number of cells that were distributed in each BIN was counted and expressed as a percentage. Scale bars, 50 μm. *P < 0.05 (Student’s t test). (B) denotes cell distribution across the cortex as a summary, while (C) shows a representative image of the mouse cortex expressing either GFP, mutant ATP1A3, or wild-type ATP1A3. (D) Left: Representative images of monitored cultured neurons introduced with wild-type or p.Asp992del ATP1A3 plasmids together with GCaMP plasmids. Regions of interest (ROIs) were marked with four different colors to monitor the intensities of GCaMP signals. Right: Graphs of intensity plot in one…

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  Fig. 8. ATP1A3-related disorders as ion channel diseases from fetal to adulthood stages. A schematic showing an overview of ATP1A3-related disorders as ion channel diseases throughout the human life span, together with other known genes, based on the latest knowledge (29). The x axis approximately (not linearly) shows the life span of a human, from newborn to adulthood. The y axis represents disease severity. MCD, malformation of cortical development; DEE, developmental and epileptic encephalopathy.