A homozygous STIM1 mutation impairs store-operated calcium entry and natural killer cell effector function without clinical immunodeficiency

Abstract

To the Editor: Stromal interaction molecule 1 (STIM1) is a transmembrane protein pivotal to store-operated calcium entry (SOCE) that localizes to either the cell or endoplasmic reticulum (ER) membranes, with the N-terminus in either the extracellular space or the ER, respectively. Plasma membrane ORAI calcium release–activated calcium modulator 1 (ORAI1) Ca2+ channels are activated by STIM1. Families previously described with recessive STIM1 mutations (MIM #612783) had life-threatening viral, bacterial, and fungal infections; developmental myopathy; hypohidrosis; and amelogenesis imperfecta (AI; generalized developmental enamel abnormalities).1, 2, 3 We investigated a consanguineous family, segregating a novel syndrome of recessive AI and hypohidrosis by using autozygosity mapping and clonal sequencing. A homozygous rare missense mutation in STIM1 (p.L74P) in the EF-hand domain was identified (see the Methods and Results sections in this article's Online Repository at www.jacionline.org). The family was re-evaluated, with particular attention paid to features associated with recessive STIM1 mutations (Table I and see Table E1, Table E2, Table E3 in this article's Online Repository at www.jacionline.org). The 2 affected cousins (18 and 11 years old, respectively) did not have overt clinical immunodeficiency. Further evaluation of their immune systems showed a normal immunoglobulin profile with an adequate specific antibody response to both nonlive (pneumococcus, tetanus and, Hib) and live (mumps, measles, and rubella) vaccinations. In addition, both subjects had detectable IgG against varicella zoster virus after a previous uncomplicated primary infection. The younger cousin was also found to have IgG against EBV viral capsid antigen, suggesting previous exposure, but neither showed any evidence of acute infection or previous exposure to cytomegalovirus. Table I Summary of the main clinical and clinical immunologic features in subjects with either homozygous or heterozygous STIM1 c.221T>C mutations Lymphocyte studies showed stable CD8 T-cell depletion in the older affected subject only. Other lymphocyte subsets, including CD4 T, natural killer (NK), and B cells, were within the normal range (Table I). However, despite normal PHA and anti-CD3 simulation responses, T-lymphocyte and NK cell SOCE was grossly abnormal, which is consistent with disruption of the Ca2+-binding EF-hand and in keeping with previous reports for recessive STIM1 mutations (see Fig E1, A, in this article's Online Repository at www.jacionline.org).1, 2, 3 The defect in NK cell SOCE was associated with impaired NK cell effector function, as shown by assays of granule exocytosis and intracellular IFN-γ production in response to K562 tumor cells (see Fig E1, B). After recently published mouse studies, which confirmed the importance of STIM1 to neutrophil SOCE and associated functions,4 we also evaluated neutrophil function. This was found to be within normal limits. Fig E1 Defective SOCE and impaired NK cell function in STIM1-Leu74Pro patients' cells. A, Calcium flux in lymphocytes after anti-CD3/anti-CD16, 1 μmol/L thapsigargin, or 500 nmol/L ionomycin administration. B, Granule exocytosis ... Despite abnormal immune system SOCE, the affected subjects in this case appear to be able to compensate for this deficit and avoid overt immunodeficiency. It is possible that the relative preservation of T-cell function might compensate for NK cell dysfunction. Neither might yet have encountered a pathogen that would expose this particular immune system limitation (see Table E2). An ability to mount a partial response to viral infections was reported in a family with clinical immunodeficiency and a history of viral infections caused by a homozygous missense R429C change affecting the STIM1 cytoplasmic domain.2 A mouse model characterized by conditional knockout of Stim1 and Stim2 in both CD4+ and CD8+ T cells has recently provided further insight into the importance of Stim1 in immune system development and virus-specific memory and recall responses, which prevent acute viral infections from becoming chronic.5 Recessive STIM1 mutations can be associated with other immune dysregulations, including autoimmune disease. The older cousin had a transient episode of idiopathic thrombocytopenic purpura when 2 years old that might have been unrelated to the STIM1 mutations. There were no other clinical or serologic markers consistent with autoimmune disease, and regulatory T-cell numbers were normal. Both cousins were intolerant of warm environments and aware of their inability to sweat normally. This limited the older cousin's ability to participate in sport. There was no clinical or serologic evidence of myopathy. This is in contrast to other recessive STIM1 mutations and also to dominant STIM1 mutations affecting the EF-hand that cause tubular aggregate myopathy (MIM #160565).6 Hypomineralized AI affected the primary and secondary dentitions of both affected cousins (see Fig E2 in this article's Online Repository at www.jacionline.org), which is in keeping with reports of other recessive STIM1 mutations. The cousins were physically small (height, weight, and head circumference <0.4th percentile) when assessed at 18 years and 9 years, 10 months of age, respectively. Without comparable data from other subjects with recessive STIM1 mutations, it is unclear whether this is a cosegregating feature. Fig E2 Hypomineralized AI as the presenting feature in a family with STIM1 L74P change. A, Pedigree of the consanguineous family investigated. The 2 affected cousins with AI and hypohidrosis are shaded black. Genotypes of the c.221T>C variant are indicated ... The L74P STIM1 change within the EF-hand domain precedes the first Ca2+-binding aspartate residue by 2 amino acids (see Fig E2) and therefore might be expected to distort the Ca2+-binding region of the protein. Therefore we compared the response of mutant YFP-STIM1 (L74P) with the depletion of Ca2+ stores after thapsigargin or cyclopiazonic acid (CPA) treatment with that of wild-type YFP-STIM1 and the previously published EF-hand mutant7 YFP-STIM1 (D76A, see Fig E3 in this article's Online Repository at www.jacionline.org). Fig E3 STIM1 localization and Ca2+ flux in cells transfected with STIM1 constructs. A, TIRFM of HEK293 cells transfected with either wild-type (WT), D76 A mutant, or L74P mutant YFP-STIM1 after treatment with 2 μmol/L thapsigargin ... Using total internal reflection fluorescence microscopy (TIRFM), we replicated previous observations that wild-type YFP-STIM1 relocalizes to puncta proximal to the plasma membrane after treatment of transfected HEK293 cells with 2 μmol/L thapsigargin to deplete ER Ca2+ stores through sarcoendoplasmic reticulum calcium transport ATPase (SERCA) inhibition (see Fig E3, A). The EF-hand mutant YFP-STIM1 (D76 A) was present in these puncta before thapsigargin treatment, with no observable response to thapsigargin (see Fig E3, A). Similarly, mutant YFP-STIM1 (L74P) showed no response to thapsigargin but also appeared to form constitutive puncta, which was less distinct in appearance than that for the D76A mutant (see Fig E3). We compared Ca2+ fluctuations in HEK293 cells transfected with ORAI-CFP and either wild-type YFP-STIM1, mutant YFP-STIM1 (D76A), or mutant YFP-STIM1 (L74P; see Fig E3, B and C). Both YFP-STIM1 (D76A) and YFP-STIM1 (L74P) transfected cells had increased basal Ca2+ concentrations compared with wild-type YFP-STIM1 and reduced peak and integral responses to CPA-induced SERCA inhibition (see Fig E3, B and C). However, in contrast to the EF-hand mutant YFP-STIM1 (D76A), YFP-STIM1 (L74P) did not demonstrate reduced SOCE after CPA washout and Ca2+ restoration, suggesting that the previously reported desensitization of SOCE observed with the YFP-STIM1 (D76A) mutant does not occur with the YFP-STIM1 (L74P) mutant form. Therefore the L74P mutation appears to result in a distinct molecular phenotype compared with the loss of function observed in immunodeficient patients and the constitutive activation observed in patients with myopathy. This study is the first to report recessive STIM1 mutations in patients presenting with AI and hypohidrosis without overt clinical immunodeficiency or myopathy. Clinical immunologic investigations were consistent with abnormal NK cell and T-lymphocyte function that might be expected to be associated with ongoing clinical immunodeficiency. However, despite severely abnormal SOCE, this was not the case in these patients. Missense mutations affecting the EF-hand can have very different clinical phenotypes with respect to the immune system, muscle, sweating, and enamel formation. This has important implications for clinical evaluation, as well as understanding the biological functions of STIM1.