Fig 2: Single point mutation in the start codon of the AMOT gene leads to an in-frame shift to a new start codon (Met92) and thereby truncated AMOT130, herein called AMOT130ΔN, which presents with elevated protein levels.(A) Schematic representation of AMOT (upper panel) and AMOT130ΔN (lower panel) genes and proteins. ABD, angiostatin binding domain; PDZ-BD, PDZ binding domain. (B) Representative Western blot indicating an approximately 10 kDa molecular weight shift of AMOT130ΔN (first lane) compared with AMOT130 and increased protein level of AMOT130ΔN in primary skin fibroblasts obtained from proband IV1 (male patient) compared with AMOT130 in a male individual acting as a control (ctrl) and a female individual acting as a control. No change in AMOT80 protein level was detected between the patient and control groups. (C) Quantification of AMOT130ΔN and AMOT130 protein levels in patient/donor–derived primary skin fibroblasts by taking GAPDH as a housekeeping gene. n = 4 independent experiments; the SD is reported. ***P < 0.001, ordinary 1-way ANOVA with Tukey’s post hoc test. (D) Representative Western blot of overexpressed AMOT130ΔN, AMOT130, or pcDNA3.1 vector backbone control in epithelial cells (MCF7). (E) Quantification of overexpressed AMOT130ΔN and AMOT130 protein levels in MCF7 cells. The housekeeping gene was GAPDH. The mean of 3 independent experiments with SD. **P < 0.01, unpaired t test. (F) Chimeric proteins scheme. (G) Representative Western blot of 3 independent experiments depicting overexpression of chimeric, AMOT130ΔN, and AMOT130 protein levels in MCF7 cells. (H) Quantification of chimeric, AMOT130ΔN, and AMOT130 protein levels of 3 independent experiments (SD reported). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ordinary 1-way ANOVA with Tukey’s post hoc test.

Fig 3: AMOT130ΔN expression and AMOT130 overexpression disrupt the cell-cell barrier integrity in hCMECs.(A) AMOT and PKCζ costaining in hCMECs without overexpression (wild-type; upper image), or after AMOT130 (middle image) or AMOT130ΔN (bottom image) overexpression. Nuclei were stained with DAPI and are shown in blue. (B and C) TEER measurement (B) and permeability assays (C) with AMOT130ΔN, AMOT130 overexpressing, or wild-type hCMECs. Cells were cultured for 4 days in Transwell plates inserted in growth medium. After completing the TEER measurement, 70 kDa FITC-dextran was added to the apical chamber of the Transwell plates for 4 hours for permeability assay. The fluorescence intensity of the collected basal medium was measured. The SD is reported. *P ≤ 0.05, **P ≤ 0.01, ordinary 1-way ANOVA, Dunnett’s multiple comparison test.

Fig 4: AMOT-linked congenital hydrocephalus presentation in the affected family.(A) The affected family pedigree. Upper-left red quadrant denotes affected by hydrocephalus. Genotype of the p.Met1Thr AMOT variant is given. (B) CT scan of the proband IV1 during shunt reinsertion at age 3 months. (C) Sequencing chromatogram of the patient and mother showing the variant in AMOT. (D) Representative scheme depicting cerebral ventricles of healthy individual designated AMOT130 (left) and of a patient with a hydrocephalus due to the p.Met1Thr variant in AMOT130ΔN (right). CSF, cerebral spinal fluid.