Article Figures & Data
Figures
- Figure 1
Flow chart of the multi-center clinical study. Patients enrolled in this study underwent multiple genetic tests to identify germline pathogenic variants. NGS, next-generation sequencing; MLPA, multiplex ligation-dependent probe amplification.
- Figure 2
Pedigrees and variants of 3 unrelated families carrying DUOX2 variants. Pedigrees of family 1(A), 2(B), and 3(C). The genotype and phenotype information are displayed, +/− represents a heterozygous nonsense (c.1588A>T; p.K530X) variant in the DUOX2 gene, and −/− represents the wild type. (D) The four DUOX2 variants in these 3 families were validated by Sanger sequencing. Their locations are depicted in a corresponding lollipop plot of DUOX2 (created with IBS) and the conserved domains of the hDuox2 protein are also shown (E).
- Figure 3
The truncated protein, hDuox2 K530, is overexpressed in adenomas. (A) Immunohistochemistry (IHC) staining of hDuox2 with an antibody targeting the N-terminal 1–100 amino acids of human Duox2 protein in the adenoma and normal tissues from a DUOX2 p.K530X carrier. The expression of non-phospho (active) β-Catenin was also detected in these 2 samples by IHC analysis. (B) The positions of the amino acid sequence recognized by 2 different anti-Duox2 antibodies are marked. (C) Immunofluorescence staining of wild-type and truncated proteins of hDuox2 in adenomas and normal tissues from a DUOX2 p.K530X carrier as well as the tumor from a DUOX2 wild-type patient. AA, amino acid.
- Figure 4
Overexpression of truncated protein, hDuox2 K530, promotes cell proliferation through the unfolded protein response. (A) Construction of the RKO cell line with stable expression of hDuox2 K530. (B) Growth curve of RKO-hDuox2 K530 and the control group. ****P < 0.0001 (C) Confocal microscopy with anti-6 × His-tag (red) and anti-calreticulin (green) in the RKO-hDuox2 K530 cell line. (D) Immunohistochemistry staining (100×) of GRP78 in the adenoma and normal tissues from a DUOX2 p.K530X carrier. EV, empty vector.
- Figure 5
Three-dimensional (3D) models of the wild-type (blue) and mutant (green) domains. (A) Superimposition of the 3D model between wild-type (blue) and mutant (green) Ferric oxidoreductase domains are displayed. A magnified view of the region containing the wild-type Arg1110 residue (yellow) and its hydrogen bonds (yellow) formed with other residues is shown in the right upper panel. The magnified view of the mutant Gln1110 residue (pink) is shown in the right lower panel. (B) The 3D models and the superimposition between wild-type (blue) and mutant (green) FAD-binding FR-type domains are displayed. Magnified views of the region containing the wild-type Leu1343 residue (yellow) and the mutant Phe1343 residue (pink) are shown in the right upper and lower panels, respectively. Note that 3 residues around the mutation site have changed directions (red arrow).
- Figure 6
Schematic diagram of truncated protein, hDuox2 K530, causing adenomatous polyposis. The diagram shows that the truncated protein, hDuox2 K530, is translated due to a nonsense variant (c.1588A>T) occurring in the DUOX2 gene. Therefore, this misfolded protein is retained in the endoplasmic reticulum and activates an unfolded protein response, which could induce abnormal cell proliferation.
Tables
Variant Function prediction Stability prediction SIFT† Mutation Taster2‡ Mutation assessor§ i-Mutant2.0¶ MUpro†† iStable‡‡ Prediction DDG value Prediction Conf. score Prediction Conf. score c.3329G > A
(p.Arg1110Gln)Deleterious Disease causing Decrease −1.02 Decrease −0.68 Decrease 0.72 c.4027C > T
(p.Leu1343Phe)Deleterious Disease causing Medium Decrease −1.23 Decrease −1 Decrease 0.87 †SIFT classifies an amino acid substitution as “deleterious” or “tolerated” according to the normalized probability. A substitution is predicted as deleterious if the score is < 0.05 and tolerated if the score is ≥ 0.05.
‡Mutation Taster2 predicts an alteration as one of 4 possible types: “disease causing,” “disease causing automatic”, “polymorphism,” and “polymorphism automatic.”
§Mutation assessor outputs the annotations of functional impact of a variant as functional (high, medium), predicted non-functional (low, neutral).
¶I-Mutant2.0 outputs free energy change value (DDG) between mutant and wild-type protein. Stability decreases if DDG < 0 and increases if DDG > 0.
††MUpro uses a confidence score between -1 and 1 to predict the effects of mutation on protein stability and measure the confidence of the prediction. A score < 0 means the mutation decreases the protein stability, and the smaller the score, the more confident the prediction. Conversely, a score > 0 means the mutation increases the protein stability, and the bigger the score, the more confident the prediction.
‡‡iStable outputs the prediction as “stability decrease” or “stability increase.”
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