The loss-of-function p.D409G mutation hindered H₂O₂production. (A) DUOX2 mRNA was found to be abundant in the thyroids by qRT-PCR. (B) H₂O₂ concentrations in mutant thyroid tissues (n = 6) were more than twofold lower than those in WT tissues (n = 6) (**P < .01). No alterations in H₂O₂ concentrations were found in the thymuses and kidneys (WT, n = 6; D409G, n = 6; P > .05). (C) H₂O₂-producing capacities in HeLa cells were evaluated by cotransfection with either WT or D409G DUOX2 porcine cDNAs in the presence of DUOXA2. The cotransfection of D409G DUOX2 with DUOXA2 resulted in only 50% of the H₂O₂ being produced compared with WT (**P < .01; the data presented are the means ± standard error [SE] of 3 independent experiments). (D) The H₂O₂ release triggered by WT and D409G mutant DUOX2 was completely blocked by incubation with DPI. *P < .05, **P < .01; the data presented are the means ± SE of 3 independent experiments. (E) The potential structural impact of the D409G mutation (arrow) on DUOX2 was predicted by the 3D protein structure using SWISS-MODEL and HOPE. The negatively charged aspartic acid is converted into neutral glycine. (F) Protein stability assays with CHX chase showed a more rapid decrease in the amount of D409G protein in comparison with WT protein at the examined time points after 0.5 hours. (G) Western blot signals were measured using ImageJ software, and obtained values of hemagglutinin (HA)/β-actin from 3 replications were graphed to produce degradation curves (*P < .05, **P < .01). In addition to the specific notes, all data are presented as the means ± SD. DPI, diphenyleneiodonium; NS, not significant; RFU, relative fluorescence unit.