ORAI1 deficiency and lack of store-operated Ca2+ entry cause immunodeficiency, myopathy, and ectodermal dysplasia

Abstract

Background: Defects in the development or activation of T cells result in immunodeficiency associated with severe infections early in life. T-cell activation requires Ca2+ influx through Ca2+-release activated Ca2+ (CRAC) channels encoded by the gene ORAI1.

Objective: Investigation of the genetic causes and the clinical phenotype of immunodeficiency in patients with impaired Ca2+ influx and CRAC channel function.

Methods: DNA sequence analysis for mutations in the genes ORAI1, ORAI2, ORAI3, and stromal interaction molecule (STIM) 1 and 2, as well as mRNA and protein expression analysis of ORAI1 in immunodeficient patients. Immunohistochemical analysis of ORAI1 tissue distribution in healthy human donors.

Results: We identified mutations in ORAI1 in patients from 2 unrelated families. One patient is homozygous for a frameshift nonsense mutation in ORAI1 (ORAI1-A88SfsX25), and a second patient is compound heterozygous for 2 missense mutations in ORAI1 (ORAI1-A103E/L194P). All 3 mutations abolish ORAI1 expression and impair Ca2+ influx and CRAC channel function. The clinical syndrome associated with ORAI1 deficiency is characterized by immunodeficiency with a defect in the function but not in the development of lymphocytes, congenital myopathy, and anhydrotic ectodermal dysplasia with a defect in dental enamel calcification. In contrast with the limited clinical phenotype, we found ORAI1 protein expression in a wide variety of cell types and organs.

Conclusion: Ca2+ influx through ORAI1 is crucial for lymphocyte function in vivo. Despite almost ubiquitous ORAI1 expression, the channel has a nonredundant role in only a few cell types judging from the limited clinical phenotype in ORAI1-deficient patients.

Figure 1. ORAI1 A88SfsX25 nonsense mutation abolishes ORAI1 expression in P4

A, Adenosine insertion (258–259insA) results in frame shift, premature termination (A88SfsX25) and altered amino acid sequence (red) of TM1 in ORAI1 (shaded). B–C, Non-detectable ORAI1 mRNA (B) and protein (C) expression in P4 compared to control. D, Impaired Ca²⁺ influx in fibroblasts from P4 (left) is restored by expression of ORAI1 (middle). Bar graphs represent averages of peak [Ca²⁺]_i from 4–6 experiments. TG, thapsigargin.

Figure 2. Two missense mutations abolish ORAI1 protein expression in P6

A, ORAI1 C308A and T581C mutations result in single amino acid substitutions A103E and L194P in TM1 and TM3 (shaded or boxed) of ORAI1, respectively. B–C, Undetectable endogenous (B) and ectopic (C) expression of ORAI1 protein in fibroblasts from P6 (B) and HEK293 cells transfected with ORAI1 mutants (C). WT, wild-type; glyc., glycosylated. D, Impaired Ca²⁺ influx in nontransfected (nt) fibroblasts from P6 is restored by expression of ORAI1. Bar graphs represent averages of peak Ca²⁺ influx from 4–6 experiments. E, Location of ORAI1 mutations in P4 and P6.

Figure 3. EDA and myopathy in ORAI1 deficient patients

A, Normal hair in P6 (age 16y). B–D, Hypocalcified amelogenesis imperfecta with significant loss of enamel substance in deciduous (B, P2 at age 6y) and permanent (C, P2 at age 10.5y; D, P6 at age 9.5y) teeth. E–H, Atrophy of type II muscle fibers in P2 (age 5y) by ATPase (E), NADH (F) and αORAI1(red)/αMHC fast (green) staining (G-H). I–J, ORAI1 expression in normal human skeletal muscle. Pep, blocking peptide.

Figure 4. Almost ubiquitous ORAI1 tissue expression

A, ORAI1 expression in human CD4⁺, CD8⁺ and CD19⁺ T and B cells analyzed by flow cytometry. B–P, ORAI1 expression in tissues from healthy donors incubated with αORAI1 antibody. Shown are thymus (B–D), spleen (E), eccrine sweat glands (F), skin (G), adrenal gland (H), parathyroid gland (J), exocrine pancreas (K), pancreatic islet (L), liver (M), lung (N), kidney (O), cerebellum (P). Specificity controls are shown in Figure E3. A, artery; BV, blood vessel; PALS, periarterial lymphoid sheath.