De novo missense variants in phosphatidylinositol kinase PIP5KIγ underlie a neurodevelopmental syndrome associated with altered phosphoinositide signaling

Abstract

Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an in vivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants.

Keywords: PIP5K1C; de novo gain-of-function variants; developmental delay; endosomes; intellectual disability; phosphatidylinositol 4,5 bisphosphate (PI(4,5)P(2)); phosphoinositides; zebrafish.

Figure 1

Clinical and genetic features of PIP5K1C neurodevelopmental syndrome (A) Photos of I-1, I- 2, I-4, I-5, I-6, I-7, and I-8. Frequent facial features include prominent midface with protruding upper lip; broad nose with wide alae; hypertelorism; wide arched eyebrows; and ears that are long, posteriorly rotated, and have thickened superior helices. I-4 also has bilateral ptosis. (B) Top: schematic illustration of the human PIP5KIγ protein showing the PIPK domain (blue) and the residues affected (Glu146, Tyr205, Tyr221) in our cohort and described by Narkis et al. (Asp253). Bottom: amino acid sequence alignment for various PIP5KIγ vertebrate orthologs in the region of interest. Orthologs include H. Sapiens (NP_036530.1), P. troglodytes (XP_512274.3), M. mulatta (XP_002801072.1), C. lupus (XP_542172.5), B. taurus (NP_001009967.2), M. musculus (NP_032870.2), R. norvegicus (NP_001009967.2), G. gallus (XP_418191.4), D. rerio (XP_005171520.1 and XP_0051637202.1), and X. tropicalis (NP_001120474.1). Gray residues vary from the human reference. Green residues are those affected by the variants described here (Glu146, Tyr205, Tyr221), and the residue in light blue (Asp253) is the one described by Narkis et al.

Figure 2

Mutant PIP5KIƳ results in upregulation of PI(4,5)P2 levels and affects the PI composition of EEs (A) Left: immunodetection of PI(4,5)P2 with an anti-PI(4,5)P2 antibody in control (Ctrl 1) and I-2 fibroblasts fixed and co-stained with an anti-LAMP1 antibody. Right: quantification of PI(4,5)P2 levels (the ratio of PI(4,5)P2 and LAMP1 fluorescence intensity) in I-2 fibroblasts and 2 unrelated controls (Ctrl 1 and 2), expressed as % of Ctrl 1. Means ± SEM, three independent experiments, n > 50 cells per experiment; Student’s t test. ^∗∗∗∗p < 0.0001. (B) Left: Airyscan confocal analysis of endogenous PI(4,5)P2 and EEA1 colocalization in I-2 fibroblasts and control (Ctrl 1). Green, PI(4,5)P2; red, EEA1. Insets show magnifications of selected areas. Scale bar, 10 μm. Right: quantification of PI(4,5)P2/EEA1 colocalization, expressed as % of Ctrl 1 (mean ± SD) in I-2 fibroblasts and controls. Three independent experiments, n > 50 cells per experiment; Student’s t test. ^∗∗∗∗p < 0.0001; ns, not significant.

Figure 3

Mutant PIP5KIƳ promotes ectopic recruitment of F-actin at EEs Left: Airyscan confocal analysis of control and I-2 fibroblasts stained with Alexa-Fluor-phalloidin to visualize F-actin and with anti-EEA1 antibody to visualize EE compartments. Insets show enlargements of the boxed areas. White arrows indicate structures positive to phalloidin and EEA1 staining. Red, phalloidin; green, EEA1. Scale bar, 10 μm. Right: quantification of amounts of F-actin associated with endosomes is expressed as % of endosomes positive for F-actin ± SD Three independent experiments, n > 50 cells per experiment; Student’s t test. ^∗∗∗∗p < 0.0001; ns, not significant.

Figure 4

Mutant PIP5KIƳ impairs Tf recycling (A) Control (Ctrl) and I-2 fibroblasts were exposed to Alexa-Fluor-488-Tf for 1 h at 4°C and then warmed to 37°C in complete medium for 5 min. Left: representative confocal image of Tf bound to the plasma membrane in I-2 and control fibroblasts (1h at 4°C). Right: the first graph shows the quantification of cell-associated A488-Tf (1 h at 4°C) expressed as percentages of bound Tf at the plasma membrane in the control. The second graph shows the quantification of the fraction of internalized/bound Tf expressed as percentages of the control. Data are mean values ± SD (n > 50 cells per experiment; three independent experiments); ^∗∗p ≤ 0.01. (B) Control (Ctrl) and I-2 fibroblasts were loaded with Alexa-Fluor-488-Tf for 1 h at 37°C (LOAD) and chased in complete medium for 5, 15, and 30 min (CHASE). Left: representative confocal images of I-2 and control fibroblasts loaded for 1 h and chased for 15 min with Alexa-Fluor-488-Tf. Right: fluorescence intensities detected in the cells after 5, 15, and 30 min of chase were quantified and expressed as percentages of the loaded Tf in the control (0 min). Data are mean values ± Sd (n > 50 cells per experiment; three independent experiments). ^∗∗∗p ≤ 0.001; ns, not significant. Scale bar, 10 μm.

Figure 5

Disease-associated PIP5K1C variants in zebrafish induce developmental defects consistent with the human disease (A) Phenotypes obtained in embryos at 2 and 3 dpf after injection of mRNAs encoding GFP (control), PIP5K1C-WT, PIP5K1C-p.Asp253Asn, PIP5K1C-p.Glu146Lys, PIP5K1C-p.Tyr205Cys, and PIP5K1C-p.Tyr221Cys mRNAs at 20 ng/μL in one-cell-stage embryos. Aberrant embryos present a spectrum of ocular abnormalities or no eyes and microcephaly. Scale bar, 50 μm. (A′) Quantification of embryos showing the different phenotypes among the surviving embryos injected with mRNAs encoding GFP (control), PIP5K1C-WT, PIP5K1C-p.Asp253Asn, PIP5K1C-p.Glu146Lys, PIP5K1C-p.Tyr205Cys, and PIP5K1C-p.Tyr221Cys mRNAs. Total number of injected embryos per column is 222, 235, 206, 401, 157, and 173, respectively. (B) Alcian blue staining of 3-dpf larvae injected with mRNAs encoding GFP (control), PIP5K1C-WT, PIP5K1C-p.Tyr205Cys, PIP5K1C-p.Tyr221Cys, and PIP5K1C-p.Glu146Lys mRNAs at 20 ng/μL showing aberrant head structures and WT phenotype. Scale bar, 150 μm. (B′) Quantification of the distance between Meckel’s and ceratohyal cartilages (measure 1) and the ceratohyal angle (measure 2). X axis: (1) PIP5K1C-WT, WT-like embryos (n = 11); (2) PIP5K1C-p.Glu146Lys, WT-like embryos (n = 72); (3) PIP5K1C-p.Glu146Lys, embryos with abnormalities (n = 50); (4) PIP5K1C-p.Tyr205Cys, WT-like embryos (n = 15); (5) PIP5K1C-p.Tyr205Cys, embryos with abnormalities (n = 47); (6) PIP5K1C-p.Tyr221Cys, WT-like embryos (n = 13); (7) PIP5K1C-p.Tyr221Cys, embryos with abnormalities (n = 20). Wilcoxon-Mann-Whitney test: ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; n.s., not significant. (C) Left: schematic representation of the cytidine base editor technology. Middle: phenotypes obtained in the embryos mutated in pip5k1ca for the insertion of the p.Glu151Lys variant. Scale bar, 50 μm. Right: quantification of the embryos showing ocular abnormalities and microcephaly in control embryos (injected with CBE4max-SpRYmRNA, n = 186) and in the embryos injected for the insertion of the p.Glu151Lys variant (injected with CBE4max-SpRYmRNA +pip5k1ca sgRNA2, n = 215).