Identification of human NFATC1 mutations and molecular analysis of the impact of identified variants on protein stability, localization, function, and interactions. (A) Pedigree of multigenerational family; shaded symbols, patients who are affected with compound heterozygous variants in NFATC1; open symbols, heterozygous carriers, individuals with wild-type genes or not genotyped. Double lines indicate consanguinity. Crossed symbols designate deceased individuals. The genotype of the patients and family members is indicated below each symbol. The asterisk represents those individuals who underwent WES. (B) Chest radiograph and thoracolumbal radiograph showing scoliosis in patients P1 and P2, respectively (red arrows). (C) Illustration of the NFATc1 protein, with N-terminal regulatory domain (Rel homology domain [RHD] and transactivation domain A [TAD-A]), for DNA binding to the consensus sequence, and C-terminal transactivation domain (TAD-B). Location of variants are shown together with their conservation across several species. An asterisk (∗) indicates fully conserved; a colon (:), highly conserved; and a period (.), weakly conserved residues. (D) Immunoblot showing the NFATc1 expression in T cells from P1 and P2 (the patients whose material we could access) and HCs. (E) Representative blot for protein stability analysis using cycloheximide (CHX) chase assay on HEK cells transfected with either empty vector or Strep-HA-NFATc1^WT, -NFATc1^S454L, -NFATc1^V745M, or -NFATc1^V745M+S454L plasmids, followed by 6 and 10 hours of CHX treatment. Quantifications of NFATc1 expression from blots represented in panels D and E are provided in Supplemental Figure 2B and D, respectively. (F) Representative images acquired with Image Flow Cytometer (AMNIS) (left), and similarity score quantification using IDEAS software (right) of NFATc1 nuclear translocation in feeder-expanded T lymphoblasts from patients or HCs, either without stimulation (Unstim) or upon stimulation with soluble anti-CD3/CD28 antibodies (Stim). Data show significant nuclear translocation in HC cells upon stimulation and no or slight nuclear translocation in T cells from patients P1 and P2, respectively. Representative images for different similarity scores (measure of nuclear translocation) in HCs are shown adjacent to the violin plot. Cells were stained with the nuclear marker DAPI (4′,6-diamidino-2-phenylindole) (blue); NFATc1 phycoerythrin is displayed in magenta. (G) Quantification of NFATc1 activation measured as absorbance at 450 nm using an enzyme-linked immunosorbent assay–based TF activation assay on feeder-expanded T lymphoblasts from patients or HCs after stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. All experiments are representative of at least 3 independent experiments with similar findings, with the exception of unstimulated fraction shown in panel G in which n = 1. All data are mean ± standard error of the mean (SEM) and were analyzed using 2-way analysis of variance (ANOVA) with Bonferroni post hoc test in panel F and one-way ANOVA with Bonferroni post hoc test to correct for multiple comparison in panel G. Scale bar, 5 μm; original magnification ×60 for panel F.