Vibratory Urticaria Associated with a Missense Variant in ADGRE2 Genetics Journal Club 4/28/2016 Jinbo Fan PhD Fellow, Clinical Cytogenetics Division of Genomic Diagnostics The Children’s Hospital of Philadelphia

Physical Urticarias NLRP3 PLCG2 Example of Dermographism Example of Cold Urticaria Immunol Allergy Clin North Am. 2014 Feb;34(1):73-88.

Degranulations of Mast Cells A cellular process that releases a mixture of compounds, including histamine, serotonin, etc., from secretory vesicles called granules found inside the mast cells. https://en.wikipedia.org/wiki/Degranulation

A Rare Hereditary Vibratory Urticaria Ped. 3 Ped. 1 Ped. 2

Forearm Vortex Challenge

Serum Histamine Levels

Skin Biopsy: Immunohistochemical Staining of Tryptase

Genome Wide Linkage Analysis of Families 1 and 2 Genotyped ~300K SNPs across genome using Human CytoSNP-12 array (Illumina); Multipoint parametric linkage analysis was conducted by using MERLIN software;

Linkage Analysis Results: part 1 Linkage observed on 19p13 LOD = Log10 of odds Chr 19 meiotic distance vs physical distance LOD LOD Nature. 2004 Apr 1;428(6982):529-35.

Linkage Analysis Results: part 2 Family 1: A maximum LOD score of 2.1 within a 6.4 Mb region; Family 2: A maximum LOD score of 5.1 within a partial overlapping 2.2 Mb region; The combined LOD score is 7.2; An overlapping 1.7 Mb region with a shared haplotype between Families 1 and 2; Consistent with inheritance from a common ancestor.

Exome Sequencing Study of Families 1 Seven affected and three unaffected; Filtering criteria: 1) Nonsynonymous, in 5’ untranslated region, or in splice sites (6-bp); 2) Absent from dbSNP v129; 3) <0.1% frequency in ~8000 genomes/exomes from various projects; 4) Within the consensus linkage interval on 19p13; 5) Cosegregated with phenotype among family members; 3 eligible variants

Validation of Variants Identified by Exome Sequencing Study Sanger sequencing of 3 eligible variants among all available members of three families; Results: 1) One false positive call; 2) One variant not found in Family 2 (???); 3) One missense substitution, c.1475G>A (p.C492Y), perfectly co-segregated with urticaria in all available members of three families;

ADGRE2:c.1475G>A Is Not Found in dbSNP v146 database As well as from 1105 unaffected Lebanese and 100 unaffected Israeli controls.

ADGRE2 Gene Variants Screening in Various Urticaria Patients No additional rare coding or splice variants were identified in a panel of 60 patients with various sporadic physical urticarias (unrelated to each other and to families 1-3). Genetic heterogeneity of vibratory urticaria!

ADGRE2 Protein Domains Structure Cleavage between p.517-p.518 Noncovalently bound

Expression and Processing of Non-mutant and Mutant ADGRE2 ADGRE2 mRNA or protein was expressed in various human tissues (especially in primary human mast cells) (Fig. S3 A-C) Expression, cleavage, adhesion, and trafficking of mutant ADGRE2 are similar as non-mutant ADGRE2 (Fig. S4 A-F)

Vibration-Induced Degranulation of Mast Cells

Vibration-induced Subunit Dissociation NO vibration After vibration GFP (GREEN) tagged ADGRE2 beta subunit; 2A1 mono-clonal antibody (RED) targeted ADGRE2 alpha subunit;

Vibration-induced Subunit Dissociation P.C492Y substitution destabilizes the noncovalent binding between the alpha and beta subunits

Constitutive Degranulation An N-terminal truncation mutant (expressing only beta subunit) elicited constitutive degranulation Suggesting that the extracellular alpha subunit inhibits signaling by the beta subunit

Cleavage-Dependent, Vibration-Induced Degranulation Non-mutant is somewhat sensitive to vibration p.C492Y mutant is hypermorphic Cleavage is required for degranulation Transfected LAD2 cells

In Summary A novel missense variant in ADGRE2 is linked with autosomal dominant vibratory urticaria. This mutation effects a pathogenic gain of function mechanism that destabilizing the inhibitory interaction between the alpha and beta subunit. Exaggeration of a normal cellular response to dermal vibration.