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CRAC Channelopathies Due to Mutations in ORAI1 and STIM1 

CRAC Channelopathies Due to Mutations in ORAI1 and STIM1
Chapter:
CRAC Channelopathies Due to Mutations in ORAI1 and STIM1
Author(s):

Stefan Feske

DOI:
10.1093/med/9780195389838.003.0020
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Introduction

Ca2+ signals contribute to the function of many immune cells, including T and B cells, natural killer (NK) cells, mast cells, dendritic cells, and macrophages. These signals control diverse functions ranging from differentiation, proliferation, gene expression, and cell motility to secretion of vesicles containing cytokines, cytotoxic, or proinflammatory proteins (Feske, 2007). The main mechanism controlling Ca2+ influx in lymphocytes is store-operated Ca2+ entry (SOCE) through the so-called calcium release activated calcium (CRAC) channel. The importance of Ca2+ influx through CRAC channels for immunity is highlighted by the existence of patients with a combined immunodeficiency due to a defect in SOCE, CRAC channel function, and T-cell activation (Feske et al., 1996, 2006; Le Deist et al., 1995; McCarl et al., 2009; Partiseti et al., 1994; Picard et al., 2009).

In lymphocytes, Ca2+ influx is initiated by engagement of immunoreceptors such as the TCR, BCR, or Fc receptors, resulting in the activation of signaling cascades (Fig. 20.1). Importantly, inositol 1,4,5-triphosphate (InsP3) mediates the release of Ca2+ ions from the endoplasmic reticulum (ER) and activation of stromal interaction molecule (STIM) 1. Multimerization of STIM1 in the membrane of the ER leads to the opening of the store-operated CRAC channel protein ORAI1 in the plasma membrane and sustained Ca2+ influx from the extracellular space. The term “store-operated Ca2+ entry” refers to the fact that the filling state of the ER Ca2+ store controls the opening of calcium channels in the plasma membrane. The CRAC channel is defined by its unique functional properties, measured by patch clamping, an electrophysiological method to measure ion channel currents. Both SOCE and CRAC channels represent a universal Ca2+ influx mechanism employed by lymphocytes and many other cell types. Besides CRAC channels, other ion channels are indirectly involved in the regulation of Ca2+ influx in lymphocytes. These include the nonselective cation channel TRPM4 and the potassium channels KCNN4 and KCNA3, which together control the plasma membrane potential; a negative membrane potential is required to promote passive influx of Ca2+ ions through open CRAC channels and along the electrochemical gradient. Thus, theoretically mutations in a number of genes may affect Ca2+ levels in lymphocytes, including molecules operating proximal to InsP3 production and ER store depletion as well as those affecting SOCE more directly, such as STIM1, ORAI1, potassium, and TRPM channels.

Figure 20.1 Store-operated Ca2+ entry (SOCE) through ORAI1 and STIM1 in T cells. (A) Following T-cell receptor (TCR) stimulation, the intracellular Ca2+ concentration [Ca2+]i rises from ~50–100 nM at rest to ~1 µM. This increase in [Ca2+]i following the activation of tyrosine kinases Lck and ZAP-70 (ζ‎-chain-associated protein kinase of 70 kDa), phosphorylation of adaptor proteins such as SLP76 (SH2-domain-containing leukocyte protein of 76 kDa) and LAT (linker for activation of T cells), and activation of phospholipase (PLC) γ‎1. PLCγ‎1 hydrolyses phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) in the plasma membrane to inositol-1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 binds to and opens InsP3 receptors (InsP3Rs) in the endoplasmic reticulum (ER), resulting in transient efflux of Ca2+. Lower ER Ca2+ sensed by stromal interaction molecule 1 (STIM1) leads to the opening of calcium-release activated calcium (CRAC) channels in the plasma membrane, which are composed of ORAI1 subunits. Sustained Ca2+ influx through CRAC channels activates calcineurin and the transcription factor nuclear factor of activated T cells (NFAT). Known deleterious mutations in ORAI1 and STIM1 are indicated. STIIM1 domains: EFh, EF hand; SAM, sterile alpha motif; CC, coiled-coil. (B) Absent CRAC channel current ICRAC in T cells of an ORAI1 deficient patient (P, with mutation R91W) compared to those of a control (Ctrl), measured in whole-cell patch-clamp recordings (Feske et al, 2005). Cells in which ER Ca2+ stores had been passively depleted were subjected to voltage ramps from –100 to +50 mV. (C) Impaired SOCE in ORAI1-R91W homozygous mutant (P) vs. heterozygous parental (M, F) or control (Ctrl) T cells. Single-cell ionized calcium concentration, [Ca2+]i, was measured by time-lapse microscopy after T-cell loading with the Ca2+ indicator dye Fura-2 and stimulation with thapsigargin (TG, arrow) to passively deplete ER Ca2+ in the absence of extracellular Ca2+ (open bar). After re-addition of Ca2+ (black bar), SOCE was undetectable in patient T cells and markedly reduced in heterozygous T cells.

Figure 20.1
Store-operated Ca2+ entry (SOCE) through ORAI1 and STIM1 in T cells. (A) Following T-cell receptor (TCR) stimulation, the intracellular Ca2+ concentration [Ca2+]i rises from ~50–100 nM at rest to ~1 µM. This increase in [Ca2+]i following the activation of tyrosine kinases Lck and ZAP-70 (ζ‎-chain-associated protein kinase of 70 kDa), phosphorylation of adaptor proteins such as SLP76 (SH2-domain-containing leukocyte protein of 76 kDa) and LAT (linker for activation of T cells), and activation of phospholipase (PLC) γ‎1. PLCγ‎1 hydrolyses phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) in the plasma membrane to inositol-1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 binds to and opens InsP3 receptors (InsP3Rs) in the endoplasmic reticulum (ER), resulting in transient efflux of Ca2+. Lower ER Ca2+ sensed by stromal interaction molecule 1 (STIM1) leads to the opening of calcium-release activated calcium (CRAC) channels in the plasma membrane, which are composed of ORAI1 subunits. Sustained Ca2+ influx through CRAC channels activates calcineurin and the transcription factor nuclear factor of activated T cells (NFAT). Known deleterious mutations in ORAI1 and STIM1 are indicated. STIIM1 domains: EFh, EF hand; SAM, sterile alpha motif; CC, coiled-coil. (B) Absent CRAC channel current ICRAC in T cells of an ORAI1 deficient patient (P, with mutation R91W) compared to those of a control (Ctrl), measured in whole-cell patch-clamp recordings (Feske et al, 2005). Cells in which ER Ca2+ stores had been passively depleted were subjected to voltage ramps from –100 to +50 mV. (C) Impaired SOCE in ORAI1-R91W homozygous mutant (P) vs. heterozygous parental (M, F) or control (Ctrl) T cells. Single-cell ionized calcium concentration, [Ca2+]i, was measured by time-lapse microscopy after T-cell loading with the Ca2+ indicator dye Fura-2 and stimulation with thapsigargin (TG, arrow) to passively deplete ER Ca2+ in the absence of extracellular Ca2+ (open bar). After re-addition of Ca2+ (black bar), SOCE was undetectable in patient T cells and markedly reduced in heterozygous T cells.

ORAI1 Deficiency

Biology of ORAI1

The CRAC channel gene ORAI1 (or CRACM1, TMEM142a) on human chromosome 12q24 is the founding member of a new class of ion channels structurally related to ORAI2 (CRACM2, TMEM142b) and ORAI3 (CRACM3, TMEM142c). ORAI1 has only two exons and encodes a highly conserved 301 amino acid protein with a tetra-spanning plasma membrane topology. ORAI1 is the pore-forming subunit of the CRAC channel. A negatively charged glutamate residue, E106, in its first transmembrane domain functions as a Ca2+ binding site in the ion channel pore (Prakriya et al., 2006; Vig et al., 2006a; Yeromin et al., 2006). CRAC channels are thought to be tetramers, with each ORAI1 subunit contributing a glutamate residue for coordinated Ca2+ binding in the CRAC channel pore. ORAI2 and ORAI3, which share the predicted tetra-spanning membrane topology with ORAI1, can form Ca2+ channels when ectopically expressed in vitro, and it is possible that endogenous ORAI2 and ORAI3 play a role in CRAC channel function and SOCE in immune cells.

Molecular Basis of ORAI1 Deficiency

Discovery of ORAI1 by Modified Linkage Analysis and RNAi Screens

ORAI1 was discovered in three independent genome-wide RNAi screens of drosophila S2 cells for regulators of Ca2+ signaling and activation of the transcription factor NFAT (Feske et al., 2006; Vig et al., 2006b; Zhang et al., 2006) and by positional cloning (Feske et al., 2006). The latter approach relied on a strategy of genome-wide linkage analysis in a pedigree with only two patients by (1) identifying potential heterozygous disease carriers through functional tests in vitro (i.e., magnitude of Ca2+ influx) and (2) combining two independent modes of haplotype analysis. Twelve of 21 relatives of the patients showed ~50 percent reduced Ca2+ influx in their T cells compared to controls, suggesting that they were heterozygous carriers of the gene defect (Fig. 20.1C). Their DNA was used for microarray-based genome-wide single-nucleotide-polymorphism (SNP) mapping followed by evaluation of SNP data in two independent linkage analyses for an autosomal recessive and dominant disease trait, respectively (Feske et al., 2006). Together, both linkage analyses yielded a combined LOD score of 5.7, defining a 6.5 Mb interval on chromosome 12q24 containing ~74 genes. The hypothetical gene locus FLJ14466 located in the candidate region was sequenced because its drosophila homolog olf-186F was among the positive hits in a RNAi screen for NFAT activating genes (Feske et al., 2006). FLJ14466 and olf-186F were renamed ORAI1 and dOrai, respectively, after the Orai (hours) Eunomia (Harmony), Dyke (Justice), and Eirene (Peace), the keepers of heaven’s gate in Greek mythology (Stewart, 2005).

Mutations in ORAI1

To date, three families with six patients have been reported to lack CRAC channel function and SOCE due to mutations in ORAI1 (Fig. 20.1A, Table 20.1). An R91W missense mutation in exon1 of ORAI1 was identified in two patients from the first family whose T cells lacked SOCE and ICRAC (Fig. 20.1B, C). Both patients were homozygous for a C→T transition at position 271 of the ORAI1 coding sequence (NM_032790), substituting highly conserved arginine residue 91 with tryptophan at the beginning of the first transmembrane domain of ORAI1(Feske et al., 2006). Hydrophobicity of the mutant tryptophan at position 91 is essential to abolish channel function; replacement of R91 with lipophilic leucine, phenylalanine, or valine, but not with charged or neutral amino acids, impaired CRAC channel function (Derler et al., 2009; McCarl et al., 2009).

Table 20.1 Phenotypes of ORAI1 and STIM1 Deficiency

ORAI1

STIM1

Chromosome

12q24

11q15

Gene defects (all autosomal recessive)

R91W, A88EfsX25, A103E/L194P

E128RfsX9

# of patients (families)

6 (3)

3 (1)

Clinical Symptoms

Immunodeficiency

Viral, bacterial, fungal infections

Viral, bacterial, fungal infections

Autoimmunity

Neutropenia (1 patient)

Autoimmune hemolytic anemia,

thrombocytopenia,

lymphoadenopathy, hepatosplenomegaly

Congenital myopathy

Global muscular hypotonia,

atrophic type II muscle fibers (R91W),

respiratory insufficiency (R91W,

A103E/L194P)

Global muscular hypotonia

Ectodermal dysplasia

Enamel dentition defect: Amelogenesis imperfecta type III (R91W),

anhydrosis

Enamel dentition defect

Other

Idiopathic encephalopathy

Facial dysmorphy

Nephrotic syndrome (1 patient)

Outcome

Death (in first yr): 4/6

Survival after HSCT: 2/6

Death (1.5–9 yrs): 2/3

Survival after HSCT: 1/3

Laboratory Findings—Immunological

Lymphocyte counts

Normal

Normal

T, B, NK cell subsets

Normal

Normal,

except CD4+ CD25+ Foxp3+ Treg ↓

T cell activation (in vitro)

Proliferation ↓↓

Cytokines (IL-2, IL-4, IFN-γ‎) ↓↓

Proliferation ↓-↓↓

Immunoglobulins

Normal—↑ Ig levels,

no seroconversion

Normal Ig levels,

no seroconversion

Laboratory Findings—Signaling

Protein expression

Yes (R91W), no (A88EfsX25),

no (A103E/L194P)

No

SOCE/ICRAC

Absent/absent

Absent/not tested

ORAI1 and STIM1 deficiency is characterized by a defect in T-cell activation resulting in immunodeficiency, congenital myopathy, and anhydrotic ectodermal dysplasia. Lack of STIM1 expression in addition is associated with hepatosplenomegaly and autoimmunity due to greatly reduced numbers of Treg cells. For details see text.

HSCT, hematopoietic stem cell transplantation; ICRAC, Ca2+ release activated Ca2+ (CRAC) channel current; SOCE, store-operated Ca2+ entry.

A patient from a second family (Fig. 20.1A), born to consanguineous parents, lacked SOCE and CRAC channel activity (Partiseti et al., 1994) and was homozygous for insertion of a single adenine between nucleotides 258 and 259 (258insA) of the ORAI1 coding sequence (McCarl et al., 2009). This mutation at the end of the first transmembrane domain caused a frameshift at amino acid 88 and a premature termination codon at position 112 (ORAI1 A88EfsX25) (Fig. 20.1A). The lack of ORAI1 mRNA and protein in this patient is consistent with nonsense-mediated mRNA decay.

Two missense mutations in ORAI1 exon 2, A103E and L194P, were identified in an affected patient from a third family (Le Deist et al., 1995; McCarl et al., 2009) (Fig. 20.1A). Both mutations interfered with stable protein expression as no ORAI1 protein was detected in the patient’s fibroblasts or in HEK293 cells ectopically expressing these ORAI1 mutants. SOCE could be reconstituted in T cells and fibroblasts from all ORAI1-deficient patients by retroviral transduction with expression vectors encoding wild-type ORAI1 (Feske et al., 2006; McCarl et al., 2009).

Clinical and Immunological Phenotype of ORAI1 Deficiency

The dominant clinical phenotype in all patients was immunodeficiency, with severe infections early in life, but they also had congenital myopathy and ectodermal dysplasia. Recurrent severe infections were due to viral, bacterial, mycobacterial, and fungal pathogens causing pneumonia, meningitis, enteritis, gastrointestinal candidiasis, and sepsis in the various patients (Table 20.1) (Feske et al., 1996, 2000; Le Deist et al., 1995; McCarl et al., 2009; Partiseti et al., 1994). Antibiotics and intravenous immunoglobulin (IVIg) only inefficiently controlled infections, necessitating hematopoietic stem cell transplantation (HSCT). Two of six patients were treated successfully by HSCT, but the remaining four patients died in their first year of life. ORAI1-deficient patients resembled patients with severe combined immunodeficiency (SCID), although lymphocyte counts and numbers of CD4+ and CD8+ T cells and of B cells were normal. T-cell activation was severely compromised, with impaired proliferation and cytokine production in response to TCR-dependent and independent stimuli in vitro and absent skin delayed-type hypersensitivity reactions in vivo (Table 20.1) (Feske et al., 1996, 2000; Le Deist et al., 1995; Partiseti et al., 1994; Schlesier et al., 1993). Despite the activation defect in vitro, increased numbers of T cells with an activated (CD3+ HLA-DR+) and memory (CD4+CD45RO+, CD4+ CD29+) phenotype were observed in the peripheral blood of all patients analyzed for these markers.

ORAI1-deficient patients also developed early global muscular hypotonia with decreased head control, delayed ambulation, and reduced muscle strength and endurance (McCarl et al., 2009). The two surviving patients after HSCT had hypotonia of respiratory muscles, chronic pulmonary disease, superinfections,bronchiectasis by the time of adolescence. Histologically, the myopathy was characterized by a variation in muscle fiber size, with a predominance of type I fibersatrophic type II fibers (Plate 20.1). Other structural abnormalities commonly found in congenital myopathies were not observed.

Plate 20.1 Dental and muscle abnormalities in patients with ORAI1-deficiency. Dental enamel defects are shown in A, B and on radiogram, C; muscle fiber abnormalities shown in D, E include a predominance of type I fibers and atrophic type II fibers.

Plate 20.1
Dental and muscle abnormalities in patients with ORAI1-deficiency. Dental enamel defects are shown in A, B and on radiogram, C; muscle fiber abnormalities shown in D, E include a predominance of type I fibers and atrophic type II fibers.

Ectodermal dysplasia with anhydrosis (EDA), impaired sweat production,a defect in dental enamel formation occurred in both surviving ORAI1-deficient patients (Plate 20.1). Dry skinheat intolerance led to recurrent fevers. Hypocalcified dental enamel matrix led to use-dependent loss of the soft enamelpainful exposure of underlying dentin, consistent with the diagnosis amelogenesis imperfecta type III (Plate 20.1). Scalp hair and eyebrows, often sparse or missing in other forms of EDA, were normal in ORAI1-deficient patients.

Encephalopathy observed in a patient with a ORAI1 A88EfsX25 nonsense mutation and his unaffected brother was judged unlikely to be due to ORAI1 deficiency because it occurred in only one of three families with ORAI1 defects (Partiseti et al., 1994).

STIM1 Deficiency

Biology of STIM1

The human STIM1 gene on chromosome 11p15 consists of 12 exons. STIM1 is a single-pass transmembrane protein of 685 amino acids localized predominantly in the membrane of the ER, where it functions as a sensor of ER Ca2+ concentrations and activator of ORAI1/CRAC channels (Liou et al., 2005; Roos et al., 2005). STIM1 contains a pair of low-affinity EF hand calcium-binding domains, a sterile alpha motif (SAM), and two coiled-coil protein–protein interaction domains. Depletion of Ca2+ from the ER results in dissociation of Ca2+ from the N-terminal EF hand domains of STIM1, unfolding of the EF-SAM domain, and multimerization of STIM1, ultimately leading to the assembly of STIM1 in large ER membrane clusters called puncta (Liou et al., 2007; Stathopulos et al., 2008). The formation of STIM1 puncta causes aggregation of ORAI1 in the plasma membrane and localized Ca2+ influx (Liou et al., 2005, 2007; Luik et al., 2006, Wu et al., 2006). STIM2, a closely related paralog of STIM1, is also located in the ER, is able to heterodimerize with STIM1, and acts as a positive regulator of SOCE (Manji et al., 2000; Williams et al., 2001). STIM2 activates Ca2+ influx upon smaller decreases in ER Ca2+ concentrations than STIM1 and was shown to regulate basal cytosolic Ca2+ concentrations (Brandman et al., 2007).

Molecular Basis of STIM1 Deficiency

To date, three patients from one family have been reported to lack SOCE due to mutations in STIM1 (Picard et al., 2009). Patient fibroblasts showed a pronounced defect in SOCE in response to thapsigargin, an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), which induces passive depletion of intracellular Ca2+ stores. Born to consanguineous parents, the proband and her younger brother were homozygous for insertion of an adenine in exon 3 of STIM1, resulting in a frameshift beginning at codon 128 and a premature termination codon at position 136 (E128RfsX9). Consequently, STIM1 mRNA and protein were greatly reduced or absent in the patients’ cells. SOCE was restored by retroviral transduction with wild-type STIM1 while partial reconstitution was achieved by expression of STIM2, suggesting that the genes have overlapping functions. Endogenous expression levels of STIM2 were not, however, sufficient to compensate for the lack of STIM1 in the patients.

Clinical and Immunological Phenotype of STIM1 Deficiency

Lack of STIM1 is characterized by immunodeficiency, congenital myopathy, and ectodermal dysplasia reminiscent of ORAI1-deficient patients but in addition results in autoimmune disease (Table 20.1) (Picard et al., 2009). Patients had recurrent bacterial and viral infections such as urinary tract infections, bacterial sepsis, otitis media, and pneumonia caused by a spectrum of pathogens, including S. pneumoniae, E. coli, cytomegalovirus, and varicella zoster virus. Lymphocyte counts were slightly reduced or normal. The proband had an age-appropriate distribution of lymphocyte subpopulations and normal TCR repertoire but decreased proportions of naïve CD4+ T cells and CD4+CD45RA+CD31+ T cells (recent emigrants from the thymus). T-cell proliferation in response to stimulation with phytohemagglutinin, phorbol 12-myristate 13-acetate plus ionomycin, or anti-CD3 antibody was markedly impaired, with even less response to recall antigens.

Immunodeficiency in the STIM1-deficient patients was complicated by hepatosplenomegaly and autoimmune disease (Picard et al., 2009). Two patients had lymphadenopathy and hepatosplenomegaly, but Fas-induced T-cell apoptosis measured in one was normal. All had thrombocytopenia and two had autoimmune hemolytic anemia. A likely cause for autoimmunity in STIM1-deficient patients is their reduced number of CD4+ CD25+ FoxP3+ regulatory T cells (Treg). Mice lacking expression of STIM1 and STIM2 also show Treg cell defects (Oh-Hora et al., 2008). Lack of SOCE and reduced TCR signal strength in developing T cells may cause abnormal persistence of self-reactive T cells during thymic development, leading to the autoimmunity in human patients and mice. Despite reduced numbers of Treg cells, STIM1-deficient patients did not resemble patients with X-linked immune dysregulation, polyendocrinopathy, enteropathy (IPEX) syndrome (Ochs et al., 2007), presumably because of impaired antigen-specific activation of effector T cells in the absence of STIM1 and SOCE.

Like ORAI1-deficient patients, those lacking STIM1 also suffered from ectodermal dysplasia and congenital myopathy as well as partial iris hypoplasia. The myopathy is consistent with the role of STIM1 in myoblast differentiation and the defect in skeletal muscle development and function found in Stim1-deficient mice (Darbellay et al., 2008; Lyfenko and Dirksen, 2008; Stiber et al., 2008).

Animal Models

Gene-targeted mice lacking Orai1 and Stim1 expression have been generated by homologous recombination and insertional mutagenesis. In contrast to ORAI1- and STIM1-deficient patients (Feske et al., 2006; Picard et al., 2009), mice generally die in the first days postpartum, most likely due to hypotonia. Surviving Orai1-/- and Stim1-/- mice are severely runted but may catch up with their littermates in the first weeks of life, depending on genetic background. Stim1-/- mice show morphological abnormalities in skeletal muscle and defects in myoblast function.

Immune function is compromised in Orai1-/- and Stim1-/- mice due to severely impaired CRAC channel function and SOCE in CD4+ and CD8+ T cells, B cells, mast cells, and macrophages (Baba et al., 2008; Gwack et al., 2008; Oh-Hora et al., 2008), although one study found only mild impairment of Ca2+ influx in Orai1-deficient T cells (Vig et al., 2008). As a consequence, expression of cytokines interleukin (IL)-2, interferon (IFN)-γ‎, IL-4, and IL-10 was substantially reduced in T cells from Orai1-/- and Stim1-/- mice (Gwack et al., 2008; Oh-Hora et al., 2008), similar to ORAI1-deficient human patients (Feske et al., 2000). By contrast, T-cell proliferation in response to TCR stimulation and T-cell-dependent antibody responses were preserved in Orai1-/- (Gwack et al., 2008) and Stim1-/- mice (Beyersdorf et al., 2009). By contrast, B cells from Orai1-/- mice proliferated poorly in response to BCR stimulation (Gwack et al., 2008), and mast cells from both Orai1-/- and Stim1-/- mice showed reduced cytokine secretion and degranulation in vitro and attenuated passive cutaneous anaphylaxis in vivo (Baba et al., 2008; Vig et al., 2008). Finally, macrophages lacking Stim1 expression had severely compromised FcRγ‎II/III-mediated Ca2+ influx (Braun et al., 2008) and were protected from disease in in vivo models of autoantibody-mediated thrombocytopenia and anemia. These findings suggested a role for SOCE in phagocytosis in mice (Braun et al., 2008), in contrast to autoimmune hemolytic anemia and thrombocytopenia in STIM1-deficient patients (Picard et al., 2009).

Autoimmunity, lymphadenopathy, and splenomegaly observed in STIM1-deficient patients was recapitulated in mice with conditional, T-cell-specific deletion of both Stim1 and Stim2 (Stim1f/f, Stim2f/f CD4-Cre). These mice had reduced numbers and impaired function of Treg cells (Oh-Hora et al., 2008). In addition, double-deficient mice showed leukocytic organ infiltration, colitis, dermatitis, and blepharitis. A potential cause for the paucity of Treg cells is the failed Ca2+-dependent activation of NFAT, which interacts with binding sites in the promoter and enhancer of FoxP3, the lineage-determining transcription factor of Treg cells (Tone et al., 2008).

Myeloid- and lymphoid-cell development in the bone marrow and thymus was unperturbed in Orai1-, Stim1-, and Stim1/Stim2-deficient mice, consistent with the normal leukocyte numbers in ORAI1- and STIM1-deficient patients (Beyersdorf et al., 2009; Feske et al., 1996; Gwack et al., 2008; Le Deist et al., 1995; McCarl et al., 2009; Oh-Hora et al., 2008; Partiseti et al., 1994; Picard et al., 2009). Ca2+ signals are widely considered necessary for differentiation and selection of T cells in the thymus, but T-cell development was normal in Stim1-deficient mice (Stim1f/f CMV-Cre) despite a complete lack of detectable SOCE (M. Oh-Hora, A. Rao, SF unpublished); these data suggest that STIM1 and ORAI1 may be dispensable for lymphocyte development, with the notable exception of Treg cells.

Prognosis and Treatment

Despite normal lymphocyte development in patients lacking functional ORAI1 or STIM1, the immunodeficiency is similar in scope and severity, especially in ORAI1-deficient patients, to that of SCID patients. Four of six ORAI1-deficient patients died in their first year of life due to recurrent, severe infections, and two STIM1-deficient patients died at 1.5 and 9 years of life of encephalitis and complications of HSCT, respectively. HSCT resulted in successful immune reconstitution in two ORAI1-deficient patients and one STIM1-deficient patient. The two now-adolescent ORAI1-deficient patients, however, suffer from secondary complications of muscular hypotonia and chronic pulmonary disease. In addition, one developed a monoclonal EBV-associated polymorphic B-cell lymphoma of host origin at 8 years of age. No signs of autoimmunity were observed in the surviving STIM1-deficient patient after HSCT.

Concluding Remarks

The clinical phenotypes of ORAI1 and STIM1 deficiency largely overlap, suggesting that the developmental and functional defects are not protein-specific but rather pathway-specific—that is, that they result from the absence of SOCE and CRAC channel function. The immunodeficiency in both diseases is caused by a severe defect in T-cell activation but not T-cell development. However, STIM1, but not ORAI1, seems required for the development of CD4+ Foxp3+ regulatory T cells, evidenced by the reduced numbers of Treg cells in the peripheral blood of one STIM1-deficient patient and autoimmune lymphoproliferative disease in all three patients (Picard et al., 2009). While numbers of Treg cells could not be evaluated in ORAI1-deficient patients, symptoms of autoimmunity were apparent in only one of the patients, who had neutropenia and thrombocytopenia at 7 months of age (McCarl et al., 2009). As ORAI1-deficient patients either died in their first year of life and or were treated by HSCT, it can be speculated that a defect in Treg development and subsequent autoimmunity did not have enough time to manifest in these patients. Alternatively, residual SOCE in immature T cells in ORAI1-deficient (in contrast to STIM1-deficient) patients may permit Treg development. Given the expression of ORAI2 in naïve CD4+ T cells, it is conceivable that other ORAI isoforms such as ORAI2 or ORAI3 play a role in SOCE in immature T cells and that mutations in these genes may be associated with defects in T-cell development or function.

Finally, the novel strategy used to positionally clone the ORAI1 gene described in this chapter may be useful for the identification of gene defects underlying other rare autosomal recessive diseases in which traditional linkage analysis cannot yield high enough LOD scores due to the small number of affected patients. Successful application of this approach relies on (1) a reliable test to identify heterozygous carriers and (2) a large enough number of relatives who can be tested.

Acknowledgments

This work was supported by grants from the March of Dimes Foundation, the Charles H. Hood Foundation, and the NIH. I would like to thank Drs. A. Rao and A. Fischer for their support.

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