240 - Dominant-negative NOX5 variant linked to primary immunodeficiency disorder

Abstract

Several NOX enzymes have been linked to immunodeficiencies and inflammatory diseases. NOX5, a superoxide-generating NOX enzyme originally detected in the spleen, testes, and lymph nodes, is the least understood member of the NOX/DUOX family of NADPH oxidases, since the gene is absent in mice and rats, and no human diseases have been linked to NOX5. Using whole-exome sequencing of patients with immunodeficiencies, we identified an in-frame, three-codon deletion in NOX5. Here, we explored the functional consequences of this mono-allelic NOX5 variant (NOX5.P323_G325) on NADPH oxidase activity in reconstituted cell models. The alignment of this NOX5 sequence with other NOX isozymes shows that the 3-codon deletion occurs within the fourth transmembrane helix, between heme-binding helices 3 and 5. While predicted to be highly disruptive, western blotting for NOX5 detects an ~75 kDa band in both WT NOX5 and NOX5.P323_G325 transfected cells, with variant stability similar to WT NOX5. In comparison to WT NOX5-transfected cells, cells transfected with the NOX5.P323_G325 variant produced significantly less superoxide when stimulated with ionomycin, PMA, or H2O2 in all three cell models. Furthermore, the mutated protein causes dose-dependent inhibition of NADPH oxidase activity of co-expressed wild-type NOX5 in two NOX5-reconstituted models (COS-7 and HEK 293 cells), providing further insight on dominant-negative effects of this monoallelic gene variant. Taken together, our findings represent the first case in which a genetic and functional defect in NOX5 has been linked to immunodeficiency and raise the question of NOX5 involvement in lymphoid cell dysfunction.