Figure 1. Nonsense Mutation in the Stromal Interaction Molecule 1 Gene (STIM1) and Lack of Store-Operated Ca2+ Entry into Cells.
Panel A shows the pedigree of the proband with combined immunodeficiency disease (Patient V-1) and her affected siblings (Patients V-4 and V-7). The STIM1 genotypes of patients and family members from whom DNA was available for sequence analysis (all of whom were in generation IV or V) are listed under their symbols; no DNA was available for Patient V-4. WT denotes wild type. Squares indicate male family members, circles female family members, solid symbols patients, open symbols unaffected persons, slashes deceased persons, and double horizontal lines consanguinity. Panel B shows intracellular calcium levels measured in fibroblasts from Patient V-1 and from a control, showing a reduction of Ca2+ influx in fibroblasts from Patient V-1. Depletion of Ca2+ stores was induced with thapsigargin (TG) in Ca2+-free Ringer’s solution, followed by perfusion with Ringer’s solution containing 20 mM Ca2+ to allow for Ca2+ influx. The resultant intracellular Ca2+ levels were measured as the ratio of fluorescence emissions at 510 nm of Fura-2, a fluorescent dye that binds calcium, after excitation at 340 nm and 380 nm, respectively. Between 40 and 80 cells per experiment were analyzed; results of one representative experiment are shown in the plots on the left side, in which the horizontal bars indicate the durations of perfusion with Ca2+-containing (black) and Ca2+-free (white) Ringer’s solution and the duration of stimulation with thapsigargin (gray). The plots to the right show the average peak Ca2+ levels and initial Ca2+ influx rates after the perfusion of 20 mM Ca2+ (corrected for the baseline values, which were those before the initial depletion of the Ca2+ stores) from four independent experiments. I bars represent the standard error. Panel C shows the results of sequence analysis of genomic STIM1 DNA obtained from Patient V-1 and from a control, revealing an insertion of a single adenine in STIM1 exon 3, between positions 380 and 381 of the coding sequence (380_381insA; NM_003156) resulting in a frame shift and premature termination codon (X) at position 136 of the protein (E136X; NP_003147). The structures of the STIM1 protein domains are superimposed on the sequence of STIM1 exons (modified from Williams et al.15) in Panel D, showing the location of the E136X truncation mutation in STIM1 exon 3, resulting in a truncated STIM1 protein in the N-terminal end of its sterile alpha-motif (SAM) domain. The abbreviation cEF denotes canonical EF-hand domain, hEF hidden EF-hand domain (as recently identified by Stathopulos et al.16), K lysine-rich domain, S/P serine–proline-rich domain, TM transmembrane domain, and UTR untranslated region.