Functional Domain Mapping of TPO: Insights From 6 Variants in Sudanese Kindreds With Congenital Hypothyroidism

Abstract

Context: Congenital hypothyroidism (CH) is a leading cause of preventable intellectual disability worldwide if left untreated. Thyroid peroxidase (TPO) is a key enzyme that uses hydrogen peroxide from the DUOX/DUOXA system to oxidize iodide for thyroid hormone synthesis.

Objective: This work aimed to identify the pathogenic TPO variants responsible for CH.

Methods: Variants identified by whole-exome sequencing were analyzed using in silico tools and structural modeling for pathogenicity. TPO function was assessed through in vitro studies on intracellular trafficking, enzymatic activity, and interaction with DUOX/DUOXA proteins.

Results: Six TPO variants were identified: p.G395D, p.V618M, p.M706V, and p.T725P in family 1, and p.R648G and p.G771R in families 2 and 3, respectively. Affected individuals in family 1 exhibited compound heterozygous or homozygous variants for the 4 variants. In silico analyses showed incomplete concordance in predicting pathogenicity. In vitro studies confirmed p.G395D as the primary pathogenic variant in family 1, and p.R648G and p.G771R in families 2 and 3. Notably, p.V618M, p.M706V, and p.T725P did not impair TPO function, either individually or in combination, suggesting that these regions are not critical for enzymatic activity. Further functional analyses revealed that p.G771R is essential for proper membrane insertion, whereas p.R648G is necessary for enzymatic activity.

Conclusion: When multiple TPO variants occur within the same family, a combination of in silico and in vitro analyses can help identify the variant responsible for the phenotype. In silico methods, however, cannot predict the different mechanisms of impairment, such as enzyme activity vs cellular localization, where the protein's topology is essential for normal function.

Keywords: DUOXA; DUOXA2; congenital hypothyroidism; dyshormonogenesis; thyroid peroxidase.

Figure 1.

Schematic representation of the biochemical reactions involved in thyroid hormone (TH) biosynthesis. Summary of the key molecular components and enzymatic steps in TH biosynthesis, emphasizing the role of thyroid peroxidase (TPO). DUOX2 with its maturation factor DUOXA2 produces hydrogen peroxide (H₂O₂), the essential oxidant for TPO activity. TPO catalyzes the oxidation of iodide and its incorporation into tyrosine residues of thyroglobulin in a process known as organification, leading to the formation of monoiodotyrosine (MIT) and diiodotyrosine (DIT). TPO then mediates the oxidative coupling of iodotyrosines to synthesize the TH triiodothyronine (T3) and thyroxine (T4). Illustration produced by Sapiens and used with permission.

Figure 2.

Phenotype and genotype of family 1. Generations are denoted by roman numerals. Each individual is identified by a number above and to the right of each symbol. Thyroid function tests are aligned below each symbol. Abnormal values are in bold and underlined. Iodine status was not available for any individuals at the time of evaluation. Abbreviations: FT4, free thyroxine; TBG, thyroxine-binding globulin; TG, thyroglobulin; TG Ab, antithyroglobulin antibody; TPO Ab, anti-TPO antibody; TSH, thyrotropin; TT3, total triiodothyronine; TT4, total thyroxine; UK, unknown.

Figure 3.

Phenotype and genotype of A, family 2 and B, family 3. Generations are denoted by roman numerals. Each individual is identified by the number above and to the right of each of each symbol. Thyroid function tests are aligned below each symbol. Abnormal values are in bold and underlined. Iodine status was not available for any individuals at the time of evaluation. Abbreviations: FT4, free thyroxine; TBG, thyroxine-binding globulin; TG, thyroglobulin; TG Ab, anti-thyroglobulin antibody; TPO Ab, anti-TPO antibody; TSH, thyrotropin; TT3, total triiodothyronine; TT4, total thyroxine; UK, unknown.

Figure 4.

Overview of 3-dimensional wild-type (WT) and variant–thyroid peroxidase (TPO) proteins, close view structures from the same angle, all of which were generated using SWISS-MODEL. It indicates the location of each TPO variant and amino acid changes. A, Four variants identified in family 1; B, variant in family 2; C, variant in family 3.

Figure 5.

Functional analysis of thyroid peroxidase (TPO) variants identified in family 1 (p.G395D, p.V618M, p.M706V, p.T725P). A, Schematic representation of the TPO variants identified in family 1. B, Western blot analysis under reducing conditions, showing the protein expression levels of each TPO variant in HEK293 cells. β-Actin served as the loading control. C, Immunofluorescence images of wild-type (WT) and the 4 TPO variants, with TPO stained in green and DAPI (4′,6-diamidino-2-phenylindole)–stained nuclei in blue. D, Western blot analysis of TPO expression on the cell surface and in the flow-through fraction. Cells expressing WT TPO without biotin served as a negative control. E, Enzymatic activity of WT and the 4 TPO variants, measured using the Amplex Red assay with extracellular hydrogen peroxide (H₂O₂) generated by glucose oxidase-catalyzed glucose oxidation. F, Enzymatic activity of WT and variant TPOs measured using the Amplex Red assay with H₂O₂ generated by cotransfected DUOXA2/DUOX2. Values are expressed as mean ± SEM. ****P value less than .0001. Illustration produced by BioRender and used with permission.

Figure 6.

Functional analysis of the variant thyroid peroxidase (TPO) (p.R648G) identified in family 2. A, Schematic representation of the variant TPO identified in family 2. B, Western blot analysis under reducing conditions, showing the expression levels of the TPO variant protein in HEK293 cells. β-Actin served as the loading control. C, Immunofluorescence images of the p.R648G variant, with TPO stained in green and DAPI (4′,6-diamidino-2-phenylindole)–stained nuclei in blue. D, Western blot analysis of TPO expression on the cell surface and in the flow-through fraction. Cells expressing WT TPO without biotin served as a negative control. E, Enzymatic activity of wild-type (WT) TPO and the p.R648G measured using the Amplex Red assay with extracellular hydrogen peroxide (H₂O₂) generated by glucose oxidase-catalyzed glucose oxidation. F, Enzymatic activity of WT TPO and p.R648G measured using the Amplex Red assay with H₂O₂ generated by cotransfected DUOXA2/DUOX2. Values are expressed as mean ± SEM. ****P value less than .0001. Illustration produced by BioRender and used with permission.

Figure 7.

Functional analysis of the variant thyroid peroxidase (TPO) identified in family 3 (p.G771R). A, Schematic representation of the variant TPO identified in family 3. B, Western blot analysis under reducing conditions, showing the expression levels of the p.G771R variant protein in HEK293 cells. β-Actin served as the loading control. C, Immunofluorescence images of the p.G771R variant, with TPO stained in green and DAPI (4′,6-diamidino-2-phenylindole)–stained nuclei in blue. D, Western blot analysis of TPO expression on the cell surface and in the flow-through fraction. Cells expressing wild-type (WT) TPO without biotin served as a negative control. E, Enzymatic activity of WT and p.G771R TPOs measured using the Amplex Red assay with extracellular hydrogen peroxide (H₂O₂) generated by glucose oxidase-catalyzed glucose oxidation. F, Enzymatic activity of WT and p.G771R TPOs measured using the Amplex Red assay with H₂O₂ generated by co-transfected DUOXA2/DUOX2. Values are expressed as mean ± SEM. **P value less than .01 and ****P value less than .0001. Illustration produced by BioRender and used with permission.