Hypothyroidism-associated missense mutation impairs NADPH oxidase activity and intracellular trafficking of Duox2

In the thyroid gland Duox2-derived H₂O₂ is essential for thyroid hormone biosynthesis. Several patients were identified with partial or severe iodide organification defects caused by mutation in the gene for Duox2 or its maturation factor, DuoxA2. A Duox2-deficient (Duox2^thyd) mouse model enabled in vivo investigation of its critical function in thyroid tissues, but its roles proposed in host defense or other innate responses in nonthyroid tissues remain less certain. These mice carry a spontaneous DUOX2 missense mutation, a T→G transversion, in exon 16 that changes the highly conserved valine 674 to glycine and results in severe congenital hypothyroidism. The exact mechanism underlying the effects of the V674G mutation has not been elucidated at the molecular or cellular level. To determine how the V674G mutation leads to congenital hypothyroidism, we introduced the same mutation into human Duox2 or Duox1 cDNAs and expressed them in HEK-293 cells stably expressing the corresponding DuoxA proteins. We found that the valine→glycine mutant Duox proteins fail to produce H₂O₂, lose their plasma membrane localization pattern, and are retained within the endoplasmic reticulum. The Duox2 mutant binds to DuoxA2, but appears to be unstable owing to this retention. Immunohistochemical staining of Duox2 in murine salivary gland ducts showed that Duox2 in mutant mice loses its condensed apical plasma membrane localization pattern characteristic of wild-type Duox2 and accumulates in punctate vesicular structures within cells. Our findings demonstrate that changing the highly conserved valine 674 in Duox2 leads to impaired subcellular targeting and reactive oxygen species release required for hormonogenesis, resulting in congenital hypothyroidism.