The broad phenotypic spectrum of PPP2R1A-related neurodevelopmental disorders correlates with the degree of biochemical dysfunction

Abstract

Purpose: Neurodevelopmental disorders (NDD) caused by protein phosphatase 2A (PP2A) dysfunction have mainly been associated with de novo variants in PPP2R5D and PPP2CA, and more rarely in PPP2R1A. Here, we aimed to better understand the latter by characterizing 30 individuals with de novo and often recurrent variants in this PP2A scaffolding Aα subunit.

Methods: Most cases were identified through routine clinical diagnostics. Variants were biochemically characterized for phosphatase activity and interaction with other PP2A subunits.

Results: We describe 30 individuals with 16 different variants in PPP2R1A, 21 of whom had variants not previously reported. The severity of developmental delay ranged from mild learning problems to severe intellectual disability (ID) with or without epilepsy. Common features were language delay, hypotonia, and hypermobile joints. Macrocephaly was only seen in individuals without B55α subunit-binding deficit, and these patients had less severe ID and no seizures. Biochemically more disruptive variants with impaired B55α but increased striatin binding were associated with profound ID, epilepsy, corpus callosum hypoplasia, and sometimes microcephaly.

Conclusion: We significantly expand the phenotypic spectrum of PPP2R1A-related NDD, revealing a broader clinical presentation of the patients and that the functional consequences of the variants are more diverse than previously reported.

Keywords: PP2A; PPP2R1A; epilepsy; intellectual disability; neurodevelopmental disorder.

Fig. 1. Schematic overview of neurodevelopmental delay-associated variants in the PP2A scaffolding Aα subunit.

(a) De novo pathogenic variants in PPP2R1A identified in individuals with intellectual disability and (neuro)developmental delay are shown with respect to their exonic and Aα protein localization respectively, with exon notation by Roman numbering (I–XV) and indications of the 15 HEAT repeats (HRs) within the protein structure. (b) Detailed amino acid sequence of the affected HRs (HR1, HR4, HR5, HR6, and HR7), with the intrarepeat loop indicated in purple, and the affected amino acids underlined and in bold. (c) More schematic representation of the antiparallel helical structures of the HRs with the B subunit binding intrarepeat loops. The red crosses indicate the approximate locations of the mutated amino acids found in the affected individuals.

Fig. 2. Binding of PP2A Aα variants to representative members of all PP2A regulatory B subunit families.

(a, c–i). GFP-tagged B55α (a), B”’/STRN3 (c), B56δ (d), B56α (e), B56β (f), B56γ1 (g), B56ε (h), B”/PR72 (i) or GFP alone (as a control) was coexpressed with HA-tagged wild-type (WT) or variant Aα proteins in HEK293T cells. The presence of Aα variants was subsequently assessed in GFP traps by anti-HA immunoblotting. Representative blots can be found in Figs. S2–S9. Shown is the average value +/- SD of the ratios of the quantified anti-HA signal versus the quantified anti-GFP signal for a given Aα variant, relative to those of WT Aα (set at 100% in each experiment, dotted line), as determined in at least three independent binding experiments (n ≥ 3). A one-sample t-test (compared with 100%) was used to assess statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (b) PP2A activity measurements on a PP2A-B55-specific phosphopeptide. HA-tagged WT and variant Aα proteins were purified from transfected HEK293T cells by HA pulldown, and absolute PP2A activity was determined on the I-N-G-S-P-R-(p)T-P-R-R-G-Q-N-R phosphopeptide substrate using Biomol®Green. Specific PP2A activities were calculated by correcting the measured activities for actual Aα inputs, determined by anti-HA immunoblotting. Results represent the average specific activity +/- SD for a given Aα variant, relative to the specific activity of WT Aα (set at 100% in each experiment), as determined in at least three independent experiments (n ≥ 3). A one-sample t-test (compared with 100%) was used to assess statistical significance (*p < 0.05; **p < 0.01; ****p < 0.0001).

Fig. 3. Binding of PP2A Aα variants to the C subunit and Aα variant-associated activity.

(a) C subunit binding. HA-tagged wild-type (WT) Aα and Aα variants were purified from transfected HEK293T cells by HA pulldown, and the presence of endogenous C subunit in the complexes determined by anti-A immunoblotting. (b) Quantified C subunit binding values. Results represent the average value +/- SD of the ratios of the quantified anti-C signal versus the quantified anti-HA signal for a given Aα variant, relative to those of WT Aα (set at 100% in each experiment), as determined in at least three independent experiments (n ≥ 3). A one-sample t-test (compare to 100%) was used to assess statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (c) Associated phosphatase activity of Aα variants, measured on a nonspecific PP2A substrate. HA-tagged WT Aα and Aα variants were purified from transfected HEK293T cells by HA pulldown, and PP2A activity was determined on the artificial, holoenzyme nonspecific K-R-(p)T-I-R-R phosphopeptide substrate using Biomol®Green. Specific PP2A activities were calculated by correcting the measured activities for actual Aα inputs, determined by anti-HA immunoblotting. Results represent the average specific activity   +/- SD for a given Aα variant, relative to the specific activity of WT Aα (set at 100% in each experiment), as determined in at least three independent experiments (n ≥ 3). A one-sample t-test (compared with 100%) was used to assess statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001). (d, e) Dendritic spine analysis in hippocampal neurons expressing Aα variant p.Ser152Phe. Confocal images of fixed primary hippocampal neurons (DIV 13) 48 hours after transfection with pEGFP (negative control), WT Aα–GFP or Aα Ser152Phe-GFP (d). Quantification of the number of dendritic spines for the transiently expressed GFP, WT Aα, and Aα variant p.Ser152Phe (e). For each neuron analyzed, the number of spines in 10-μm sections was counted: pEGFP (3 transfections; 19 cells, 134 sections), Aα–GFP (3 transfections; 18 cells, 190 sections), Aα Ser152Phe-GFP (3 transfections; 18 cells, 231 sections). Each plot represents the mean value of sections analyzed in one neuron. Statistical comparative analysis was performed using a nonparametric Kruskal–Wallis test followed by Dunn multiple comparisons tests. ****p < 0.0001, ns not significant.