The Dual Oxidase Duox2 stabilized with DuoxA2 in an enzymatic complex at the surface of the cell produces extracellular H2O2 able to induce DNA damage in an inducible cellular model.

Thyroid hormone synthesis requires H2O2, produced by two NADPH oxidases, Duox1 and Duox2. To be fully active at the apical pole of the thyrocytes, these enzymes need additional maturation factors DuoxA1 and DuoxA2. The proteins have been shown to be localized at the cell surface, suggesting that they could form a complex with Duox counterparts. We have generated multiple HEK293 Tet-On3G cell lines that express various combinations of DuoxA upon doxycycline induction, in association with a constitutive expression of the Duox enzyme. We compared Duox specific activity, Duox/DuoxA cell surface interactions and the cellular consequences of sustained H2O2 generation. By normalizing H2O2 extracellular production by Duox or DuoxA membrane expression, we have demonstrated that the most active enzymatic complex is Duox2/DuoxA2, compared to Duox1/DuoxA1. A direct cell surface interaction was shown between Duox1/2 and both DuoxA1 and DuoxA2 using the Duolink® technology, Duox1/DuoxA1 and Duox2/DuoxA2 membrane complexes being more stable than the unpaired ones. A significant increase in DNA damage was observed in the nuclei of Duox2/DuoxA2 expressing cells after doxycycline induction and stimulation of Duox catalytic activity. The maturation and activity of Duox2 were drastically impaired when expressed with the glycosylation-defective maturation factor DuoxA2, while the impact of the unglycosylated DuoxA1 mutant on Duox1 membrane expression and activity was rather limited. The present data demonstrate for the first time that H2O2 produced by the Duox2/DuoxA2 cell surface enzymatic complex could provoke potential mutagenic DNA damage in an inducible cellular model, and highlight the importance of the co-expressed partner in the activity and stability of Duox/DuoxA complexes.

Dual Oxidase Maturation Factor 1 Positively Regulates RANKL-Induced Osteoclastogenesis via Activating Reactive Oxygen Species and TRAF6-Mediated Signaling.

Receptor activator of NF-κB ligand (RANKL) induces generation of intracellular reactive oxygen species (ROS), which act as second messengers in RANKL-mediated osteoclastogenesis. Dual oxidase maturation factor 1 (Duoxa1) has been associated with the maturation of ROS-generating enzymes including dual oxidases (Duox1 and Duox2). In the progression of osteoclast differentiation, we identified that only Duoxa1 showed an effective change upon RANKL stimulation, but not Duox1, Duox2, and Duoxa2. Therefore, we hypothesized that Duoxa1 could independently act as a second messenger for RANKL stimulation and regulate ROS production during osteoclastogenesis. Duoxa1 gradually increased during RANKL-induced osteoclastogenesis. Using siRNA or retrovirus transduction, we found that Duoxa1 regulated RANKL-stimulated osteoclast formation and bone resorption positively. Furthermore, knockdown of Duoxa1 decreased the RANKL-induced ROS production. During Duoxa1-related control of osteoclastogenesis, activation of tumor necrosis factor receptor-associated factor 6 (TRAF6)-mediated early signaling molecules including MAPKs, Akt, IκB, Btk, Src and PLCγ2 was affected, which sequentially modified the mRNA or protein expression levels of key transcription factors in osteoclast differentiation, such as c-Fos and NFATc1, as well as mRNA expression of osteoclast-specific markers. Overall, our data indicate that Duoxa1 plays a crucial role in osteoclastogenesis via regulating RANKL-induced intracellular ROS production and activating TRAF6-mediated signaling.

Homozygous DUOXA2 mutation (p.Tyr138*) in a girl with congenital hypothyroidism and her apparently unaffected brother: Case report and review of the literature.

Mutations in DUOXA2, encoding dual oxidase maturation factor 2, is a rare genetic cause of congenital hypothyroidism. Only four biallelic DUOXA2 mutation carriers have been described to date. This study was conducted to report the clinical and genetic findings of a DUOXA2 mutation-carrying family, and to review the previously reported cases. The proband was a 4-year-old girl, who was diagnosed as having congenital hypothyroidism in the frame of newborn screening. She had a high serum TSH level (138 mU/L) and a low free T4 level (0.4 ng/dL). Ultrasonography revealed goiter. She was immediately treated with levothyroxine. At age 3 years, reevaluation of her thyroid function showed a slightly elevated serum TSH level (11.0 mU/L) with normal free T4 level. Screening of the eleven congenital hypothyroidism-related genes demonstrated a previously reported nonsense DUOXA2 mutation (p.Tyr138*) in the homozygous state. Unexpectedly, we also found that the elder brother of the proband, who had no significant past medical history, had the identical homozygous mutation. Using expression experiments with HEK293 cells, we confirmed that p.Tyr138* was a loss-of-function mutation. In the literature, clinical courses of three patients were described, showing characteristic age-dependent improvement of the thyroid function. In conclusion, The proband showed comparable clinical phenotype to previously reported cases, while her brother was unaffected. The phenotypic spectrum of DUOXA2 mutations could be broader than currently accepted.

Detection of superoxide anion and hydrogen peroxide production by cellular NADPH oxidases.

BACKGROUND: The recent recognition that isoforms of the cellular NADPH-dependent oxidases, collectively known as the NOX protein family, participate in a wide range of physiologic and pathophysiologic processes in both the animal and plant kingdoms has stimulated interest in the identification, localization, and quantitation of their products in biological settings. Although several tools for measuring oxidants released extracellularly are available, the specificity and selectivity of the methods for reliable analysis of intracellular oxidants have not matched the enthusiasm for studying NOX proteins. SCOPE OF REVIEW: Focusing exclusively on superoxide anion and hydrogen peroxide produced by NOX proteins, this review describes the ideal probe for analysis of O2(-) and H2O2 generated extracellularly and intracellularly by NOX proteins. An overview of the components, organization, and topology of NOX proteins provides a rationale for applying specific probes for use and a context in which to interpret results and thereby construct plausible models linking NOX-derived oxidants to biological responses. The merits and shortcomings of methods currently in use to assess NOX activity are highlighted, and those assays that provide quantitation of superoxide or H2O2 are contrasted with those intended to examine spatial and temporal aspects of NOX activity. MAJOR CONCLUSIONS: Although interest in measuring the extracellular and intracellular products of the NOX protein family is great, robust analytical probes are limited. GENERAL SIGNIFICANCE: The widespread involvement of NOX proteins in many biological processes requires rigorous approaches to the detection, localization, and quantitation of the oxidants produced. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Dual oxidases control release of hydrogen peroxide by the gastric epithelium to prevent Helicobacter felis infection and inflammation in mice.

BACKGROUND & AIMS: Dual oxidases (DUOX) are conserved reduced nicotinamide adenine dinucleotide phosphate oxidases that produce H2O2 at the epithelial cell surface. The DUOX enzyme comprises the DUOX and DUOX maturation factor (DUOXA) subunits. Mammalian genomes encode 2 DUOX isoenzymes (DUOX1/DUOXA1 and DUOX2/DUOXA2). Expression of these genes is up-regulated during bacterial infections and chronic inflammatory diseases of the luminal gastrointestinal tract. The roles of DUOX in cellular interactions with microbes have not been determined in higher vertebrates. METHODS: Mice with disruptions of Duoxa1 and Duoxa2 genes (Duoxa(-/-) mice) and control mice were infected with Helicobacter felis to create a model of Helicobacter pylori infection--the most common human chronic infection. RESULTS: Infection with H. felis induced expression of Duox2 and Duoxa2 in the stomachs of wild-type mice, and DUOX protein specifically localized to the apical surface of epithelial cells. H. felis colonized the mucus layer in the stomachs of Duoxa(-/-) mice to a greater extent than in control mice. The increased colonization persisted into the chronic phase of infection and correlated with an increased, yet ineffective, inflammatory response. H. felis colonization also was increased in Duoxa(+/-) mice, compared with controls. We observed reduced expression of the H2O2-inducible katA gene in H. felis that colonized Duoxa(-/-) mice, compared with that found in controls (P = .0002), indicating that Duox causes oxidative stress in these bacteria. In vitro, induction of oxidative defense by H. felis failed to prevent a direct bacteriostatic effect at sustained levels of H2O2 as low as 30 µmol/L. CONCLUSIONS: Based on studies of Duoxa(-/-) mice, the DUOX enzyme complex prevents gastric colonization by H. felis and the inflammatory response. These findings indicate the nonredundant function of epithelial production of H2O2 in restricting microbial colonization.

Swelling-induced upregulation of miR-141-3p inhibits hepatocyte proliferation.

Background & Aims: MicroRNAs (miRNAs) act as a regulatory mechanism on a post-transcriptional level by repressing gene transcription/translation and play a central role in the cellular stress response. Osmotic changes occur in a variety of diseases including liver cirrhosis and hepatic encephalopathy. Changes in cell hydration and alterations of the cellular volume are major regulators of cell function and gene expression. In this study, the modulation of hepatic gene expression in response to hypoosmolarity was studied. Methods: mRNA analyses of normo- and hypoosmotic perfused rat livers by gene expression arrays were used to identify miRNA and their potential target genes associated with cell swelling preceding cell proliferation. Selected miR-141-3p was also investigated in isolated hepatocytes treated with miRNA mimic, cell stretching, and after partial hepatectomy. Inhibitor perfusion studies were performed to unravel signalling pathways responsible for miRNA upregulation. Results: Using genome-wide transcriptomic analysis, it was shown that hypoosmotic exposure led to differential gene expression in perfused rat liver. Moreover, miR-141-3p was upregulated by hypoosmolarity in perfused rat liver and in primary hepatocytes. In concert with this, miR-141-3p upregulation was prevented after Src-, Erk-, and p38-MAPK inhibition. Furthermore, luciferase reporter assays demonstrated that miR-141-3p targets cyclin dependent kinase 8 (Cdk8) mRNA. Partial hepatectomy transiently upregulated miR-141-3p levels just after the initiation of hepatocyte proliferation, whereas Cdk8 mRNA was downregulated. The mechanical stretching of rat hepatocytes resulted in miR-141-3p upregulation, whereas Cdk8 mRNA tended to decrease. Notably, the overexpression of miR-141-3p inhibited the proliferation of Huh7 cells. Conclusions: Src-mediated upregulation of miR-141-3p was found in hepatocytes in response to hypoosmotic swelling and mechanical stretching. Because of its antiproliferative function, miR-141-3p may counter-regulate the proliferative effects triggered by these stimuli. Lay summary: In this study, we identified microRNA 141-3p as an osmosensitive miRNA, which inhibits proliferation during liver cell swelling. Upregulation of microRNA 141-3p, controlled by Src-, Erk-, and p38-MAPK signalling, results in decreased mRNA levels of various genes involved in metabolic processes, macromolecular biosynthesis, and cell cycle progression.

Inhibition of the thyroid hormonogenic H2O2 production by Duox/DuoxA in zebrafish reveals VAS2870 as a new goitrogenic compound.

Thyroid hormone (TH) synthesis requires extracellular hydrogen peroxide generated by the NADPH oxidases, DUOX1 and DUOX2, with maturation factors, DUOXA1 and DUOXA2. In zebrafish, only one duox and one duoxa gene are present. Using a thyroid-specific reporter line, we investigated the role of Duox and Duoxa for TH biosynthesis in zebrafish larvae. Analysis of several zebrafish duox and duoxa mutant models consistently recovered hypothyroid phenotypes with hyperplastic goiter caused by impaired TH synthesis. Mutant larvae developed enlarged thyroids and showed increased expression of the EGFP reporter and thyroid functional markers including wild-type and mutated duox and duoxa transcripts. Treatment of zebrafish larvae with the NADPH oxidase inhibitor VAS2870 phenocopied the thyroid effects observed in duox or duoxa mutants. Additional functional in vitro assays corroborated the pharmacological inhibition of Duox activity by VAS2870. These data support the utility of this new experimental model to characterize endocrine disruptors of the thyroid function.

DUOX2 and DUOXA2 Variants Confer Susceptibility to Thyroid Dysgenesis and Gland-in-situ With Congenital Hypothyroidism.

Background: Thyroid dysgenesis (TD), which is caused by gland developmental abnormalities, is the most common cause of congenital hypothyroidism (CH). In addition, advances in diagnostic techniques have facilitated the identification of mild CH patients with a gland-in-situ (GIS) with normal thyroid morphology. Therefore, TD and GIS account for the vast majority of CH cases. Methods: Sixteen known genes to be related to CH were sequenced and screened for variations by next-generation sequencing (NGS) in a cohort of 377 CH cases, including 288 TD cases and 89 GIS cases. Results: In our CH cohort, we found that DUOX2 (21.22%) was the most commonly variant pathogenic gene, while DUOXA2 was prominent in TD (18.75%) and DUOX2 was prominent in GIS (34.83%). Both biallelic and triple variants of DUOX2 were found to be most common in children with TD and children with GIS. The most frequent combination was DUOX2 with DUOXA1 among the 61 patients who carried digenic variants. We also found for the first time that biallelic TG, DUOXA2, and DUOXA1 variants participate in the pathogenesis of TD. In addition, the variant p.Y246X in DUOXA2 was the most common variant hotspot, with 58 novel variants identified in our study. Conclusion: We meticulously described the types and characteristics of variants from sixteen known gene in children with TD and GIS in the Chinese population, suggesting that DUOXA2 and DUOX2 variants may confer susceptibility to TD and GIS via polygenic inheritance and multiple factors, which further expands the genotype-phenotype spectrum of CH in China.

Functional Characterization of DUOX Enzymes in Reconstituted Cell Models.

Biosynthesis of active human dual oxidases (DUOX1 and DUOX2) requires maturation factors, a.k.a. DUOX activator proteins (DUOXA1 and DUOXA2), that form covalent complexes with DUOX; both chains together represent the mature catalytic unit that functions as a dedicated hydrogen peroxide-generating enzyme. Genetic defects in DUOX2 or DUOXA2 can result in congenital hypothyroidism, whereas partial defects in DUOX2 activity also have been associated with very early-onset inflammatory bowel disease. Our understanding of the links between DUOX dysfunction and these diseases remains incomplete. An important challenge in developing a better understanding of the pathogenic roles of DUOX defects requires robust and reliable DUOX reconstitution cell models to examine the functional consequences of candidate DUOX missense mutations and polymorphisms. Here, we describe methods for efficient heterologous DUOX/DUOXA co-expression and functional characterization, including detailed assessments of posttranslational processing and subcellular translocation of DUOX that accompanies the maturation of these enzymes into catalytically active NADPH oxidases.

Identification of Two Missense Mutations in DUOX1 (p.R1307Q) and DUOXA1 (p.R56W) That Can Cause Congenital Hypothyroidism Through Impairing H2O2 Generation.

Context: The DUOX/DUOXA systems play a key role in H2O2 generation in thyroid cells, which is required for iodine organification and thyroid hormone synthesis. DUOX2/DUOXA2 defects can cause congenital hypothyroidism (CH), but it is unknown whether DUOX1/DUOXA1 mutations can also cause CH. Objective: We aimed to identify DUOX1/DUOXA1 mutations and explore their role in the development of CH by investigating their functional impacts on H2O2 generation. Patients and Methods: Forty-three children with CH with goiter were enrolled, in whom all exons and flanking intronic regions of DUOX1/DUOXA1 were directly sequenced. We characterized the functional effects of identified mutations on the expression of DUOX1 and DUOXA1 and H2O2 generation. Results: We identified a heterozygous DUOX1 missense mutation (G > A base substitution at nucleotide 3920 in exon 31) that changed a highly conserved arginine to glutamine at residual 1307 (p.R1307Q) in patient 1. A heterozygous-missense mutation (c.166 C>T; p.R56W) was identified in DUOXA1 in patient 2. Functional studies demonstrated that both p.R1307Q mutant or p.R56W mutant decreased the DUOX1 expression at mRNA and protein levels, with a corresponding impairment in H2O2 generation (P < 0.01). The results also showed that intact DUOXA1 was required for full activity of DUOX1 and H2O2 generation. Conclusions: We have identified two heterozygous missense mutations in DUOX1 and DUOXA1 in two patients that can cause CH through disrupting the coordination of DUOX1 and DUOXA1 in the generation of H2O2. This study for the first time demonstrates that the DUOX1/DUOXA1 system, if genetically defective, can cause CH.

DUOX Defects and Their Roles in Congenital Hypothyroidism.

Extracellular hydrogen peroxide is required for thyroperoxidase-mediated thyroid hormone synthesis in the follicular lumen of the thyroid gland. Among the NADPH oxidases, dual oxidases, DUOX1 and DUOX2, constitute a distinct subfamily initially identified as thyroid oxidases, based on their level of expression in the thyroid. Despite their high sequence similarity, the two isoforms present distinct regulations, tissue expression, and catalytic functions. Inactivating mutations in many of the genes involved in thyroid hormone synthesis cause thyroid dyshormonogenesis associated with iodide organification defect. This chapter provides an overview of the genetic alterations in DUOX2 and its maturation factor, DUOXA2, causing inherited severe hypothyroidism that clearly demonstrate the physiological implication of this oxidase in thyroid hormonogenesis. Mutations in the DUOX2 gene have been described in permanent but also in transient forms of congenital hypothyroidism. Moreover, accumulating evidence demonstrates that the high phenotypic variability associated with altered DUOX2 function is not directly related to the number of inactivated DUOX2 alleles, suggesting the existence of other pathophysiological factors. The presence of two DUOX isoforms and their corresponding maturation factors in the same organ could certainly constitute an efficient redundant mechanism to maintain sufficient H2O2 supply for iodide organification. Many of the reported DUOX2 missense variants have not been functionally characterized, their clinical impact in the observed phenotype remaining unresolved, especially in mild transient congenital hypothyroidism. DUOX2 function should be carefully evaluated using an in vitro assay wherein (1) DUOXA2 is co-expressed, (2) H2O2 production is activated, (3) and DUOX2 membrane expression is precisely analyzed.

Genetic and functional analysis of two missense DUOX2 mutations in congenital hypothyroidism and goiter.

Mutations in the dual oxidase 2 gene (DUOX2) impair hydrogen peroxide (H2O2) production and cause dyshormonogenesis. In addition, these mutations have been implicated in autosomal recessive congenital hypothyroidism (CH) with goiter. In this study, we identified DUOX2 mutations that were causative for CH and explored the effects of these mutations on DUOX2 function. Blood samples were collected from 10 infants born with CH and goiter to unrelated parents. We extracted genomic DNA and sequenced all exons by polymerase chain reaction direct sequencing. The effects of DUOX2 mutations were characterized by H2O2 production assays and cycloheximide (CHX) chase experiments. Sequence analysis revealed one novel DUOX2 mutation and one known DUOX2 mutation in unrelated families: c.1060C>T (p.R354W) and c.3616 G>A (p.A1206T). Both mutations impaired H2O2 production. CHX chase experiments demonstrated the DUOX2 mutants had shorter half-lives and degraded more rapidly than wild-type DUOX2. Our study identified two novel DUOX2 mutations in Chinese patients with CH and goiter, which were responsible for the deficit in the organification process.

Fetal Goitrous Hypothyroidism and Polyhydramnios in a Patient with Compound Heterozygous DUOXA2 Mutations.

BACKGROUND: Fetal goiter is only rarely observed in pregnant women without autoimmune thyroid disorders, and there is no epidemiological data on its pathophysiology. Dual oxidase maturation factor 2 (DUOXA2), together with dual oxidase 2, serves pivotal roles in thyroid hormone biosynthesis. To date, all reported patients with DUOXA2 mutations were diagnosed postnatally through newborn screening for congenital hypothyroidism. CASE REPORT: The mother of a male fetus presented at 33 + 4 gestational weeks (GW) with a fetal goiter and polyhydramnios. Cordocentesis revealed fetal hypothyroidism (TSH 253.4 mU/L, FT4 0.29 ng/dL). Intra-amniotic levothyroxine injections were performed at GW 34 + 3 and 35 + 3. The patient was born after spontaneous vaginal delivery at 35 + 6 GW without obstetrical complications. He was treated with levothyroxine until the age of 6 years when reevaluation of his thyroid functions showed normal results (TSH 1.32 mU/L, FT4 1.81 ng/dL). Eleven causative genes of CH, including DUOXA2, were analyzed with use of a next-generation sequencing technique. RESULTS: A next-generation sequencing-based mutation screen led us to find that he was compound heterozygous for 2 previously reported nonsense DUOXA2 mutations (p.[Tyr138*];[Tyr246*]). CONCLUSION: The present case not only illustrates the phenotypic diversity of DUOXA2 mutation carriers but also implies that DUOXA2 is important in prenatal thyroid hormone production.

Conformation of the N-Terminal Ectodomain Elicits Different Effects on DUOX Function: A Potential Impact on Congenital Hypothyroidism Caused by a H2O2 Production Defect.

BACKGROUND: Dual oxidases (DUOX1 and DUOX2) were initially identified as H2O2 sources involved in thyroid hormone synthesis. Congenital hypothyroidism (CH) resulting from inactivating mutations in the DUOX2 gene highlighted that DUOX2 is the major H2O2 provider to thyroperoxidase. The role of DUOX1 in the thyroid remains unknown. A recent study suggests that it could compensate for DUOX2 deficiency in CH. Both DUOX enzymes and their respective maturation factors DUOXA1 and DUOXA2 form a stable complex at the cell surface, which is fundamental for their enzymatic activity. Recently, intra- and intermolecular disulfide bridges were identified that are essential for the structure and the function of the DUOX2-DUOXA2 complex. This study investigated the involvement of cysteine residues conserved in DUOX1 toward the formation of disulfide bridges, which could be important for the function of the DUOX1DUOXA1 complex. METHODS: To analyze the role of these cysteine residues in both the targeting and function of dual oxidase, different human DUOX1 mutants were constructed, where the cysteine residues were replaced with glycine. The effect of these mutations on cell surface expression and H2O2-generating activity of the DUOX1-DUOXA1 complex was analyzed. RESULTS: Mutations of two cysteine residues (C118 and C1165), involved in the formation of the intramolecular disulfide bridge between the N-terminal ectodomain and one of the extracellular loops, mildly altered the function and the targeting of DUOX1, while this bridge is crucial for DUOX2 function. Unlike DUOXA2, with respect to DUOX2, the stability of the maturation factor DUOXA1 is not dependent on the oxidative folding of DUOX1. Only mutation of C579 induced a strong alteration of both targeting and function of the oxidase by preventing the covalent interaction between DUOX1 and DUOXA1. CONCLUSION: An intermolecular disulfide bridge rather than an intramolecular disulfide bridge is important for both the trafficking and H2O2-generating activity of the DUOX1-DUOXA1 complex.

DUOXA1-mediated ROS production promotes cisplatin resistance by activating ATR-Chk1 pathway in ovarian cancer.

The acquisition of resistance is a major obstacle to the clinical use of platinum drugs for ovarian cancer treatment. Increase of DNA damage response is one of major mechanisms contributing to platinum-resistance. However, how DNA damage response is regulated in platinum-resistant ovarian cancer cells remains unclear. Using quantitative high throughput combinational screen (qHTCS) and RNA-sequencing (RNA-seq), we show that dual oxidase maturation factor 1 (DUOXA1) is overexpressed in platinum-resistant ovarian cancer cells, resulting in over production of reactive oxygen species (ROS). Elevated ROS level sustains the activation of ATR-Chk1 pathway, leading to resistance to cisplatin in ovarian cancer cells. Moreover, using qHTCS we identified two Chk1 inhibitors (PF-477736 and AZD7762) that re-sensitize resistant cells to cisplatin. Blocking this novel pathway by inhibiting ROS, DUOXA1, ATR or Chk1 effectively overcomes cisplatin resistance in vitro and in vivo. Significantly, the clinical studies also confirm the activation of ATR and DOUXA1 in ovarian cancer patients, and elevated DOUXA1 or ATR-Chk1 pathway correlates with poor prognosis. Taken together, our findings not only reveal a novel mechanism regulating cisplatin resistance, but also provide multiple combinational strategies to overcome platinum-resistance in ovarian cancer.

Disorders of H2O2 generation.

After the identification of thyroid H2O2 generation system (DUOX) and of its maturation factors (DUOXA), defects in DUOX2 and/or DUOXA2 were rapidly recognized as the possible cause of congenital hypothyroidism (CH) due to thyroid dyshormonogenesis. The present Review reports data on the prevalence of DUOX2 mutations, which is variable among different series but invariably high, pointing to DUOX2 defects as one of the leading causes of dyshormonogenesis. Differently, DUOXA defects seem to be rarely involved in the pathogenesis of CH. Genotype-phenotype correlations are also reported, highlighting the great intra- and inter-familial phenotype variability which appears to be a constant feature of the defects in the H2O2 generation systems. Finally, the hypotheses to explain the phenotypic variability of the DUOX2/A2 mutations are discussed, such as the existence of other H2O2 generating systems, the age variability in thyroid hormones requirements, the differences in ethnicity, in iodine intake, and in the methodological approaches.