Phenotype of affected family members. The available family members (n = 10) consisted of 5 affected individuals (aged 4, 5, 7, 10, and 11 years at time of study), 2 healthy siblings, and 3 parents (Figure 1A). Upon clinical investigation, no abnormal symptoms were observed or reported from family members beyond anhidrosis and severe heat intolerance. In the affected family members, body growth as well as teeth, hair, nails, and skin were normal. Biochemical analysis of serum and urine in affected family members VII:4 and VII:5 (aged 11 and 10 years, respectively) revealed electrolyte levels (Na+, K+, Ca2+, Mg2+, and Cl–) within normal ranges. S-amylase levels were also normal. Starch-iodine sweat test (6) confirmed the absence of sweating in affected individuals, and all 5 exhibited abnormal increases in skin and ear canal temperature when exposed to heat (45°C, 45% humidity), accompanied by an abnormal increase in heart rate (Figure 1, B and C). Skin biopsy from the forearm of affected family member VII:4 demonstrated normal morphology and number of sweat glands.

Figure 1 Genetic analysis, clinical investigation, and InsP 3 R sequences. (A) Pedigree of the consanguineous family segregating isolated anhidrosis (black symbols). Affected individuals were homozygous for chromosome 12p marker alleles flanking ITPR2 (black bars). (B) Starch-iodine sweat test at 32°C, demonstrating sweating in a healthy control male (top) and absence of color (due to dry skin) in affected family member VII:4 with anhidrosis (bottom). Both subjects were 11 years of age. (C) Increased temperatures (left; dashed lines, skin surface; solid lines, ear canal) and heart rates (right) at rest over 25 minutes when exposed to 45°C and 45% humidity, in patients (n = 5; black lines) compared with age-matched controls (n = 3; gray lines). P < 0.05; Student’s 2-tailed t test. (D) Left: Protein sequence alignment of the pore region of InsP 3 R1–InsP 3 R3 and RYR1. Pore helix and selectivity filter are indicated. Residues of the selectivity filters are highlighted in gray, with the mutated glycine residue (p.G2498) denoted (arrow). Consensus residues correspond to amino acids conserved among all 4 proteins. Right: Interspecies alignment of the pore helix and the selectivity filter domains of InsP 3 R2 illustrated conservation of residue G2498. Data represent mean ± SD.

Genetic analysis and identification of a candidate mutation. We first performed autozygosity mapping on affected individuals (13) because of the consanguinity and the likely autosomal-recessive inheritance pattern for anhidrosis within this family. The analysis revealed a single homozygous region on chromosome 12p12.1–12p11.22 in all 5 affected individuals (Figure 1A). The region consists of 427 consecutive homozygous SNPs (rs1337853–rs2349565) spanning 31 genes over 3.4 Mb (GRCh37 25,703,471–29,137,928). Segregation of the candidate homozygous region in the family was confirmed with polymorphic microsatellite markers, and linkage analysis resulted in a maximum 2-point logarithm of odds (LOD) score of 3.08.

Targeted enrichment of the 3.4-Mb candidate region was performed on genomic DNA from affected members, followed by sequencing and filtering. The analysis identified a single novel coding variant: c.7492G>A in ITPR2 (NM_002223.2). The transition results in a predicted glycine-to-serine (p.G2498S) substitution, and it was present in a homozygous state in the 5 affected family members and in a heterozygous state in the 3 parents and 2 healthy siblings available for sampling (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI70720DS1). The glycine residue is highly conserved along the phylogenic scale from human to zebrafish (PhyloP score, 2.44645; GERP score, 4.43) among the 3 InsP 3 R subtypes as well as in the closely related ryanodine receptor 1 (RYR1; Figure 1D and ref. 10). The variant — predicted to affect protein function by PolyPhen-2 analysis (HumVar score, 1.00, probably damaging) (14) — was not found in 200 Swedish and 200 Pakistani control chromosomes, nor in 850 exomes that were available in house. Furthermore, the c.7492G>A variant is not present in the latest Exome Variant Server data release (ESP6500SI-V2; http://evs.gs.washington.edu/EVS/).

InsP 3 R immunohistochemistry in skin biopsies. Histology of a punch biopsy from the forearm of an affected family member confirmed normal morphology and number of sweat glands. Immunostaining of InsP 3 R2 was positive in the clear cells of the secretory coil of the eccrine sweat gland and was similar between control and patient samples (Figure 2, A and B). In addition, InsP 3 R2 expression was observed within the cells of the excretory duct and with a concentration in subcellular regions lining the ducts. To investigate the expression of the other InsP 3 R isoforms in eccrine sweat glands, we stained the skin biopsies for InsP 3 R1 and InsP 3 R3. Whereas InsP 3 R3 showed weak staining in the secretory part and a strong staining in the basal (peripheral) cell layer of the excretory duct (Supplemental Figure 2), we could not detect InsP 3 R1. Thus, both InsP 3 R2 and InsP 3 R3 stained positive in the secretory portion, but with somewhat complementary distributions in the excretory duct. We also stained skin biopsies for S100β, a member of the S100 protein family of Ca2+ binding proteins believed to be a secondary messenger in the Ca2+-dependent regulatory pathway for sweat secretion (15). Similar to InsP 3 R2, S100β was expressed in the clear cells of the eccrine secretory coil, but not in the dark cells or the cells of the duct (Figure 2, C and D).

Figure 2 InsP 3 R immunohistochemistry in skin biopsies. (A and B) Immunoreactivity and cellular localization of InsP 3 R2 in forearm skin biopsies of (A) a healthy control individual and (B) affected family member VII:4. Eccrine sweat glands (boxed regions) are shown enlarged. Control and patient specimens exhibited similar staining: InsP 3 R2 stained positive in the clear cells (CC), but not the dark cells (DC) (dashed lines). InsP 3 R2 was also present in cells of the excretory ducts (asterisk) with a concentration in subcellular regions lining the ducts. (C and D) Similar to InsP 3 R2, S100β staining was positive in the clear cells of the secretory coil of the eccrine sweat gland, but not in the dark cells or the cells of the duct (15), in (C) a control individual and (D) affected family member VII:4. N, nerve end. Original magnification, ×10; ×40 (enlargements). Scale bars: 20 μm.

G2498S mutation abolished the Ca2+ channel activity of InsP 3 R2. To clarify the functional consequence of the InsP 3 R2 missense mutation p.G2498S, we stably expressed WT and mutant InsP 3 R2 in DT40 chicken B lymphocytes lacking endogenous InsP 3 Rs (R23-11 cells) (16) and examined their channel properties by Ca2+ imaging. We established 3 independent cell lines expressing WT or mutated InsP 3 R2 and confirmed the expression of InsP 3 R2 protein in each stable cell line (Figure 3A). We then stimulated the cells with anti-IgM antibody to activate B cell receptors and intracellular Ca2+ release. In response to IgM stimulation, approximately 80% of cells expressing WT InsP 3 R2 showed intracellular Ca2+ oscillations (n = 139; Figure 3B), the typical form of InsP 3 R2-mediated Ca2+ release, consistent with previous studies (17). In contrast, cells expressing p.G2498S mutant InsP 3 R2 had no detectable Ca2+ response after IgM stimulation (n = 50; Figure 3B). The Ca2+ contents within the endoplasmic reticulum, as measured by passive Ca2+ release after Ca2+ pump inhibitor cyclopiazonic acid (CPA) treatment, were similar in cells expressing WT and p.G2498S mutant InsP 3 R2 (WT, 100% ± 20.72%, n = 3; p.G2498S, 129.97% ± 55.22%, n = 3; mean ± SD; P = NS, t test). These data strongly suggest that the p.G2498S mutation causes InsP 3 R2 loss of function.

Figure 3 The p.G2498S mutation abolishes the channel activity of InsP 3 R2. (A) Expression of WT and mutant p.G2498S mouse InsP 3 R2 in 3 independent stable clones. (B) Intracellular Ca2+ signals upon IgM stimulation in R23-11 cells expressing WT and p.G2498S mouse InsP 3 R2 variants. Arrows denote IgM stimulation (M4) at 0.25 μg/ml. Ca2+ signals from 2 independent p.G2498S InsP 3 R2 clones and 1 WT InsP 3 R2 clone were analyzed. Representative data (ratio change of Fura-2) from 4 independent experiments are shown. Cells expressing p.G2498S InsP 3 R2 exhibited no detectable Ca2+ signal in response to IgM stimulation (0%; n = 50 cells). Of WT InsP 3 R2 cells, 78% showed Ca2+ oscillation, 14% were Ca2+ transient, and 8% exhibited no response (n = 139 cells).

Itpr2–/– mice exhibit hypohidrosis. To further examine the contribution of InsP 3 R2 to sweat production, we examined sweat secretion in Itpr2–/– mice, which harbor a targeted disruption of Itpr2, using the starch-iodine assay (18). When pilocarpine was subcutaneously injected into the hind paws of Itpr2+/+ mice, individual sweat glands (represented by black dots) appeared within 1 minute, and the number increased in a time-dependent manner, to 78.33 ± 10.92 dots per paw at 20 minutes (mean ± SEM, n = 7; Figure 4A). In Itpr2–/– mice, however, the increase in sweat gland number was significantly attenuated (24.4 ± 2.11 dots per paw at 20 minutes, n = 5; Figure 4A). In addition, the size of each black dot (presumably representing the sweat volume from a single gland) was about half the size in Itpr2–/– versus Itpr2+/+ mice (Figure 4B). Similar to our analysis of human sweat glands, immunofluorescence staining of mouse digits confirmed InsP 3 R2 expression in S100β-expressing cells of Itpr2+/+ sweat glands, which was not seen in Itpr2–/– mice (Figure 4C).

Figure 4 Decreased sweat secretion in Itpr2–/– mice. (A) Pilocarpine-induced sweat response in Itpr2+/+ (n = 7) and Itpr2–/– (n = 5) mice visualized by the Starch-iodine assay. Representative images of Itpr2+/+ and Itpr2–/– mouse paws 20 minutes after pilocarpine injection are shown. The number of black dots (arrows) was counted at the indicated times after injection. *P < 0.05, **P < 0.005, Student’s 2-tailed t test. (B) Dot diameter 20 minutes after pilocarpine injection. (C) Immunohistochemistry of InsP 3 R2 in sweat glands of Itpr2+/+ and Itpr2–/– mice. Red, InsP 3 R2; green, S100β; blue, DAPI. Data represent mean ± SEM. Scale bar: 20 μm.

To examine Ca2+ signals in sweat glands, we dissected out individual sweat glands from mouse paws, loaded them with the Ca2+ indicators Fura Red and Fluo4, and subjected them to stimulation with various concentrations of acetylcholine. In glands from Itpr2+/+ mice, we found that the amplitude of Ca2+ signals in the secretory part increased in a dose-dependent manner (Figure 5, A and B, and Supplemental Video 1), but there were no changes in Ca2+ signals in the excretory duct (Figure 5A, arrow). We then compared the peak amplitude of Ca2+ signals in dissected Itpr2+/+ and Itpr2–/– sweat glands after acetylcholine stimulation and found an approximately 40%–50% reduction in the latter at every dose tested (Figure 5, B and C). Consistent with the reduced Ca2+ signals, Western blot analysis of sweat gland lysates with anti–pan-InsP 3 R antibody demonstrated that total InsP 3 R expression in Itpr2–/– sweat glands decreased to about 40% that of Itpr2+/+ sweat glands (Figure 5D). Thus, the extant but reduced sweat production and the residual Ca2+ signals in Itpr2–/– mouse sweat glands are likely due to expression of InsP 3 R1 and InsP 3 R3. This was further supported by the positive immunohistochemical staining for InsP 3 R1 and InsP 3 R3 in the clear cells of mouse sweat glands (Supplemental Figure 3).