Compound heterozygosity for two ORAI1 mutations reduces protein expression. (A) Pedigree of patient with ORAI1 mutation and his unrelated parents. (B) Sanger sequencing of ORAI1 gene of the patient, father, and mother. Numbers refer to nucleotide positions in the ORAI1 coding sequence (mRNA). (C) Location of the patient’s missense mutations compared with reference sequences of ORAI1 mRNA (NM_032790.4) and ORAI1 protein (NP_116179.2). (D) Locations of ORAI1 missense mutations overlaid on the membrane topology of the ORAI1 protein. (E) Flow cytometry analysis of cell surface ORAI1 protein expression in CD4⁺ and CD8⁺ T cells from the patient (Pat), his father (Fa), mother (Mo), and an HD control. Nonpermeabilized T cells were incubated with an Alexa Fluor 647–conjugated anti-human ORAI1 mAb (2C1.1) recognizing an epitope in the second extracellular loop of ORAI1 (17, 18). Representative histogram plots (left) and quantification of the mean fluorescence intensity (MFI) of ORAI1, normalized to the MFI of HD (right). Data are from three independent experiments. Statistical significance was calculated using ordinary one-way ANOVA. For CD4⁺ T cells: P = 0.0105; 0.0226; <0.0001; 0.0016; 0.0008. For CD8⁺ T cells: P = 0.0042; 0.0065; <0.0001; 0.0009; 0.0006. *P < 0.05, **P < 0.01, ***P < 0.001.