Abstract Text: A20 (TNFAIP3) is a ubiquitin editing protein that negatively regulates multiple pro-inflammatory pathways. Heterozygous TNFAIP3 mutations cause the pleomorphic immune dysregulatory disease, haploinsufficiency of A20 (HA20). These include frameshift, null, truncating, or missense mutations, all of which reduce protein stability and expression. The current gold standard assay for pathogenicity uses A20 overexpression (transient transfection), followed by expression of NF-kB luciferase activity downstream of TNFα. We hypothesized that this assay might not accurately capture the effects of missense mutations of homeostatic protein stability leading to potential underdiagnosis of HA20 in these patients. To test this hypothesis, we used the luciferase assay to test 4 frameshift/truncating variants and 4 missense variants from our cohort of 60 HA20 patients (52 are frameshift/truncating/null and 8 missense). While A20 mediated NF-kB suppression was absent in all frameshift/truncating variants, results were not affected by missense variants. We next measured A20 protein expression in peripheral blood mononuclear cells (PBMCs) from 25 healthy subjects and 25 patients with frameshift/truncating and missense variants using western blot and ELISA. A20 is sensitively detected by western and ELISA, and expression was reduced in PBMC in all patients with TNFAIP3 variants. Together these results suggest that a better preclinical diagnostic assay is needed to determine pathogenicity of TNFAIP3 mutations, especially missense variants. Future assay (western blot and ELISA) will include the measurement of WT vs mutant A20 stability in transfection models and primary cells, as well as the assessment of TNF and LPS induced NF-kB activation in primary cells.
