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

Abstract

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.

Keywords: DUOX1; DUOXA1; H2O2 generation; congenital hypothyroidism; mutation.

Figure 1

WT and mutational sequences of exon 31 in DUOX1 (A) and exon 6 in DUOXA1 (B). (A1) Arrowhead indicates homozygous G at nucleotide 3920 in normal individuals; (A2) Arrowhead indicates the heterozygous A and G at nucleotide 3920 in patient 1. (B1) Arrowhead indicates homozygous C at nucleotide 166 in normal individuals; (B2) Arrowhead indicates the heterozygous C and T at nucleotide 166 in patient 2.

Figure 2

Sequence alignment analysis of DUOX1 (A) and DUOXA1 (B) in different species. (A) Red rectangle indicates arginine at amino acid 1307of DUOX1 located in a conserved sequence of the DUOX1 protein. (B) Red rectangle indicates arginine at amino acid 56 of DUOXA1 located in a conserved sequence of the DUOXA1 protein.

Figure 3

Relative mRNA expression levels under the various indicated vector transfections. (A) DUOX1 mRNA expression. The DUOX1 mRNA level was highest in the group with both wild-type DUOX1 and DUOXA1 expression vectors, while the group with p.R1307Q and p.R56W combination had the least mRNA expression of the DUOX1 gene compared with other experimental groups except for the group with DUOXA1 only. (B) DUOXA1 mRNA expression. Groups with p.R56W mutant were far lower than other experimental groups with DUOXA1. Each column represents the mean ± SD of three independent experiments performed in triplicate. ^**P < 0.01.

Figure 4

Western blot analysis of the DUOX1 protein expression under various indicated vector transfections. (A) Representative presentation of the gel results. The DUOX1 protein level was highest in the group with both wild-type DUOX1 and DUOXA1 expression vectors, while the group transfected with both p.R1307Q mutant and p.R56W mutant had the least expression level. (B) Densitometric measurement of DUOX1 protein expression. The results are presented as the ratio of DUOX1/β-actin, corresponding to (A). Primary antibodies against DUOX1 was used at a 1:250 dilution and Anti-beta-actin, as loading control, was used at a 1:2,000 dilution. Each column represents the mean ± SD of three independent experiments performed in triplicate. ^**P < 0.01.

Figure 5

H₂O₂ generation in cells transfected with the indicated expression vectors. (A) Measurement of fluorescence intensity (535/595 nm) under various indicated vector transfections. The group transfected with both wild-type DUOX1 and DUOXA1 had the maximum fluorescence intensity. (B) Presentation of the data in nanomoles of H₂O₂. The results were transformed from fluorescence intensity using a calibration curve. Each column represents the mean ± SD of three independent experiments performed in triplicate. ^**P < 0.01.

Figure 6

Cycloheximide (CHX) chase study. (A) DUOX1 protein expression was examined at different time periods in cells transfected with WT or p.R1307Q expression vectors. (B) DUOX1 expression was quantified densitometrically as the ratio of DUOX1/β-actin. (C) Degradation percentage of DUOX1 protein in cells transfected with WT or p.R1307Q expression vectors. Each column represents the mean ± SD of three independent experiments performed in triplicate.