Vibratory Urticaria Associated with a Missense Variant in ADGRE2

Abstract

Patients with autosomal dominant vibratory urticaria have localized hives and systemic manifestations in response to dermal vibration, with coincident degranulation of mast cells and increased histamine levels in serum. We identified a previously unknown missense substitution in ADGRE2 (also known as EMR2), which was predicted to result in the replacement of cysteine with tyrosine at amino acid position 492 (p.C492Y), as the only nonsynonymous variant cosegregating with vibratory urticaria in two large kindreds. The ADGRE2 receptor undergoes autocatalytic cleavage, producing an extracellular subunit that noncovalently binds a transmembrane subunit. We showed that the variant probably destabilizes an autoinhibitory subunit interaction, sensitizing mast cells to IgE-independent vibration-induced degranulation. (Funded by the National Institutes of Health.).

Figure 1. Inheritance and Clinical Features of Vibratory Urticaria

Panel A shows the pedigrees of the three families originating from Lebanon that had autosomal dominant inheritance of vibratory urticaria. The numbered family members in the pedigrees were available for genotyping and sequencing. Squares denote male family members, circles female family members, solid symbols affected family members, and open symbols unaffected family members. Slashes indicate deceased family members, and arrows indicate the proband in each family. Panel B shows the results of the forearm vortex challenge. Pronounced hives developed in a patient after the challenge, with redness evident to either side of prominent swelling at the site of vortex contact. Panel C shows serial measurements of histamine in serum after forearm vortex challenge; substantially greater histamine release was seen in patients than in the control. Histamine levels peaked within 5 minutes after the challenge and subsided within 60 minutes. BL denotes baseline (prechallenge level). The data points indicate the means and I bars the standard error for two technical replicates. Panel D shows immunohistochemical staining of tryptase (red) in samples obtained by means of skin biopsy; this staining labels both intact and degranulated mast cells, as well as secreted granules (indicated by arrows in the postchallenge higher-magnification views) and extracellular tryptase (diffuse red staining). Release of the granular contents of mast cells after vibration was widespread in a patient sample and limited in a control sample.

Figure 2. Vibration-Induced, ADGRE2-Enhanced Degranulation of Mast Cells from Patients

Panel A shows a schematic diagram of ADGRE2 domain structure, with five epidermal growth factor (EGF)–like adhesion domains (triangles) and a central stalk in the extracellular N-terminal α subunit, the autocatalytic cleavage site between residues p.L517 and p.S518, and the seven-pass transmembrane domain in the C-terminal β subunit, depicted noncovalently bound to the α subunit. Blue bars represent disulfide bonds predicted on the basis of homology to other EGF domain–containing proteins and EGF–seven transmembrane (TM7) adhesion G-protein–coupled receptors (GPCRs). The red bar represents the location of the p.C492Y substitution. Dermatan sulfate is the endogenous ligand for ADGRE2 and binds its fourth EGF domain. The monoclonal antibody (mAb) 2A1 was raised against the stalk domain. Panels B and C show the results of assays for vibration-induced degranulation of mast cells (measured as the release of β-hexosaminidase) in Families 1 and 2. Primary mast cells (PMCs) were derived in parallel from two patients and one control member of Family 1 and allowed to adhere to plates coated with substrate as indicated, then vibrated in the presence or absence of calcium chloride, and finally assayed for degranulation (Panel B). PMCs from two patients in Family 2, PMCs from an unrelated control, and stock human mast cells (HuMCs) were also assayed (Panel C). PMCs from patients and controls were derived in parallel from CD34+ progenitors isolated from fresh whole blood, whereas HuMCs were derived from frozen CD34+ progenitors that were previously isolated from peripheral blood after granulocyte colony-stimulating factor (G-CSF) stimulation and apheresis. Data are the means and standard errors of two independent experiments, each assayed in duplicate, from cells derived from a single blood sample from either the patient or the control.

Figure 3. Cleavage-Dependent Degranulation Elicited by p.C492Y Mutant ADGRE2 Associated with a Loss of Surface α Subunit

Panel A shows confocal slice micrographs of transfected murine bone marrow–derived mast cells. As detected by immunostaining with the 2A1 antibody (red), the p.C492Y mutant α subunit, but not the nonmutant α subunit, is lost from the plasma membrane in response to vibration. In contrast, as determined by fluorescent detection of GFP (green), the GFP-tagged β subunit is retained in the membrane. Yellow indicates an overlap of the red and green signal. The scale bars indicate 5 µm. Panel B shows the significance of the decrease in colocalization. The α subunit staining evident in cells expressing CAT–GFP fusion control vector is nonspecific; however, it was confined to the cytoplasm, whereas the β subunit of ADGRE2–GFP fusion clones was located primarily in the plasma membrane. Therefore, cytoplasmic α subunit staining was masked out for the colocalization analysis (see the Methods section in the Supplementary Appendix). Each data point is the Pearson’s coefficient, measuring the correlation between the location and intensity of the α and β subunits, in the colocalized volume of a stack of high-power-field images through the depth of the cells. Data are combined from two independent experiments. There is no mouse orthologue for ADGRE2, so only transfected human ADGRE2 is detected. Panels C and D show the measurement of constitutive (Panel C) and vibration-induced (Panel D) degranulation. To measure constitutive degranulation, transfected LAD2 cells were incubated for 5 hours to allow protein expression, and nonvibrated cells were assayed. To measure vibration-induced degranulation, transfected LAD2 cells were incubated for 5 hours to allow protein expression and were adhered to dermatan sulfate–coated plates for 3 hours; parallel cultures were then either vibrated at 750 rpm for 20 minutes or not vibrated and then were assayed. In Panels C and D, the data are means and standard errors for four independent experiments, each performed in duplicate (Panel C) or triplicate (Panel D). A single asterisk denotes P<0.05, a double asterisk P<0.01, and a triple asterisk P<0.001.