1. 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

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

3. 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.

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

5. Forearm Vortex Challenge

6. Serum Histamine Levels

7. Skin Biopsy: Immunohistochemical Staining of Tryptase

8. 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;

9. 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):

10. 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.

11. 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

12. 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;

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

14. 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!

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

16. 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)

17. Vibration-Induced Degranulation of Mast Cells

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

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

20. 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

21. 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

22. 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.