The structure of human thyroglobulin.

Thyroglobulin (TG) is the protein precursor of thyroid hormones, which are essential for growth, development and the control of metabolism in vertebrates1,2. Hormone synthesis from TG occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines, and is completed by TG proteolysis3. Tyrosine proximity within TG is thought to enable the coupling reaction but hormonogenic tyrosines have not been clearly identified, and the lack of a three-dimensional structure of TG has prevented mechanistic understanding4. Here we present the structure of full-length human thyroglobulin at a resolution of approximately 3.5 Å, determined by cryo-electron microscopy. We identified all of the hormonogenic tyrosine pairs in the structure, and verified them using site-directed mutagenesis and in vitro hormone-production assays using human TG expressed in HEK293T cells. Our analysis revealed that the proximity, flexibility and solvent exposure of the tyrosines are the key characteristics of hormonogenic sites. We transferred the reaction sites from TG to an engineered tyrosine donor-acceptor pair in the unrelated bacterial maltose-binding protein (MBP), which yielded hormone production with an efficiency comparable to that of TG. Our study provides a framework to further understand the production and regulation of thyroid hormones.

Adult mouse and human organoids derived from thyroid follicular cells and modeling of Graves' hyperthyroidism.

The thyroid maintains systemic homeostasis by regulating serum thyroid hormone concentrations. Here we report the establishment of three-dimensional (3D) organoids from adult thyroid tissue representing murine and human thyroid follicular cells (TFCs). The TFC organoids (TFCOs) harbor the complete machinery of hormone production as visualized by the presence of colloid in the lumen and by the presence of essential transporters and enzymes in the polarized epithelial cells that surround a central lumen. Both the established murine as human thyroid organoids express canonical thyroid markers PAX8 and NKX2.1, while the thyroid hormone precursor thyroglobulin is expressed at comparable levels to tissue. Single-cell RNA sequencing and transmission electron microscopy confirm that TFCOs phenocopy primary thyroid tissue. Thyroid hormones are readily detectable in conditioned medium of human TFCOs. We show clinically relevant responses (increased proliferation and hormone secretion) of human TFCOs toward a panel of Graves' disease patient sera, demonstrating that organoids can model human autoimmune disease.

Maintaining the thyroid gland in mutant thyroglobulin-induced hypothyroidism requires thyroid cell proliferation that must continue in adulthood.

Congenital hypothyroidism with biallelic thyroglobulin (Tg protein, encoded by the TG gene) mutation is an endoplasmic reticulum (ER) storage disease. Many patients (and animal models) grow an enlarged thyroid (goiter), yet some do not. In adulthood, hypothyroid TGcog/cog mice (bearing a Tg-L2263P mutation) exhibit a large goiter, whereas adult WIC rats bearing the TGrdw/rdw mutation (Tg-G2298R) exhibit a hypoplastic thyroid. Homozygous TG mutation has been linked to thyroid cell death, and cytotoxicity of the Tg-G2298R protein was previously thought to explain the lack of goiter in WIC-TGrdw/rdw rats. However, recent studies revealed that TGcog/cog mice also exhibit widespread ER stress-mediated thyrocyte death, yet under continuous feedback stimulation, thyroid cells proliferate in excess of their demise. Here, to examine the relative proteotoxicity of the Tg-G2298R protein, we have used CRISPR-CRISPR-associated protein 9 technology to generate homozygous TGrdw/rdw knock-in mice in a strain background identical to that of TGcog/cog mice. TGrdw/rdw mice exhibit similar phenotypes of defective Tg protein folding, thyroid histological abnormalities, hypothyroidism, and growth retardation. TGrdw/rdw mice do not show evidence of greater ER stress response or stress-mediated cell death than TGcog/cog mice, and both mouse models exhibit sustained thyrocyte proliferation, with comparable goiter growth. In contrast, in WIC-TGrdw/rdw rats, as a function of aging, the thyrocyte proliferation rate declines precipitously. We conclude that the mutant Tg-G2298R protein is not intrinsically more proteotoxic than Tg-L2263P; rather, aging-dependent difference in maintenance of cell proliferation is the limiting factor, which accounts for the absence of goiter in adult WIC-TGrdw/rdw rats.

Thyroglobulin Interactome Profiling Defines Altered Proteostasis Topology Associated With Thyroid Dyshormonogenesis.

Thyroglobulin (Tg) is a secreted iodoglycoprotein serving as the precursor for triiodothyronine and thyroxine hormones. Many characterized Tg gene mutations produce secretion-defective variants resulting in congenital hypothyroidism. Tg processing and secretion is controlled by extensive interactions with chaperone, trafficking, and degradation factors comprising the secretory proteostasis network. While dependencies on individual proteostasis network components are known, the integration of proteostasis pathways mediating Tg protein quality control and the molecular basis of mutant Tg misprocessing remain poorly understood. We employ a multiplexed quantitative affinity purification-mass spectrometry approach to define the Tg proteostasis interactome and changes between WT and several congenital hypothyroidism variants. Mutant Tg processing is associated with common imbalances in proteostasis engagement including increased chaperoning, oxidative folding, and engagement by targeting factors for endoplasmic reticulum-associated degradation. Furthermore, we reveal mutation-specific changes in engagement with N-glycosylation components, suggesting distinct requirements for 1 Tg variant on dual engagement of both oligosaccharyltransferase complex isoforms for degradation. Modulating dysregulated proteostasis components and pathways may serve as a therapeutic strategy to restore Tg secretion and thyroid hormone biosynthesis.

Effect of BRAFV600E and TERT Promoter Mutations on Thyroglobulin Response in Patients With Distant-Metastatic Differentiated Thyroid Cancer.

OBJECTIVE: To assess the impact of serine/threonine-protein kinase B-Raf (BRAF) V600E and telomerase reverse transcriptase (TERT) promoter mutations in patients with distant-metastatic differentiated thyroid cancer (DM-DTC) based on thyroglobulin (Tg) response to radioactive iodine (RAI) therapy. METHODS: The BRAFV600E and TERT mutations in primary tumors or metastatic lymph nodes of 114 patients with DM-DTC were retrospectively examined. RAI avidity was evaluated using a posttreatment iodine-131 whole-body scan. The Tg response was dynamically assessed at a median follow-up period of 56.50 months (interquartile range, 28.43-97.98 months). RESULTS: BRAFV600E was detected in 38.6% of cases, the TERT mutation in 21.1% of cases, and both the BRAFV600E and TERT mutations in 14.9% of cases. Patients with both the mutations tended to be older at diagnosis (P < .001) and less multifocal (P = .011) and have more aggressive histologic subtypes (P = .011) and a higher Ki-67 index (P = .003). Patients with neither mutation tended to be have more RAI avidity than those with either the BRAFV600E mutation alone or both the mutations (P = .001 and .001, respectively). Patients with both the mutations exhibited a more unfavorable Tg response than those without both the mutations and those with the BRAFV600E mutation alone (P = .001 and .013, respectively). The Tg progression-free survival was shorter in patients with the TERT mutation alone than in those with neither mutation (P = .021), and it tended to be shorter when it coexisted with the BRAFV600E mutation (P < .001); however, no significant difference was observed between those with the BRAFV600E mutation alone and those with neither mutation (P = .890). CONCLUSION: The coexistence of the BRAFV600E and TERT promoter mutations synergistically induce the loss of RAI avidity and leads to an undesirable Tg response in patients with DM-DTC. The TERT promoter mutation appears to affect Tg response more than the BRAFV600E mutation.

Screening of 23 candidate genes by next-generation sequencing of patients with permanent congenital hypothyroidism: novel variants in TG, TSHR, DUOX2, FOXE1, and SLC26A7.

PURPOSE: To date, many genes have been associated with congenital hypothyroidism (CH). Our aim was to identify the mutational spectrum of 23 causative genes in Turkish patients with permanent CH, including thyroid dysgenesis (TD) and dyshormonogenesis (TDH) cases. METHODS: A total of 134 patients with permanent CH (130 primary, 4 central) were included. To identify the genetic etiology, we screened 23 candidate genes associated with CH by next-generation sequencing. For confirmation and to detect the status of the specific familial variant in relatives, Sanger sequencing was also performed. RESULTS: Possible pathogenic variants were found in 5.2% of patients with TD and in 64.0% of the patients with normal-sized thyroid or goiter. In all patients, variants were most frequently found in TSHR, followed by TPO and TG. The same homozygous TSHB variant (c.162 + 5G > A) was identified in four patients with central CH. In addition, we detected novel variants in the TSHR, TG, SLC26A7, FOXE1, and DUOX2. CONCLUSION: Genetic causes were determined in the majority of CH patients with TDH, however, despite advances in genetics, we were unable to identify the genetic etiology of most CH patients with TD, suggesting the effect of unknown genes or environmental factors. The previous studies and our findings suggest that TSHR and TPO mutations is the main genetic defect of CH in the Turkish population.

Thyroglobulin regulates the expression and localization of the novel iodide transporter solute carrier family 26 member 7 (SLC26A7) in thyrocytes.

Solute carrier family 26 member 7 (SLC26A7), identified as a causative gene for congenital hypothyroidism, was found to be a novel iodide transporter expressed on the apical side of the follicular epithelium of the thyroid. We recently showed that TSH suppressed the expression of SLC26A7 and induces its localization to the plasma membrane, where it functions. We also showed that the ability of TSH to induce thyroid hormone synthesis is completely reversed by an autocrine negative-feedback action of thyroglobulin (Tg) stored in the follicular lumen. In the present study, we investigated the potential effect of follicular Tg on SLC26A7 expression and found that follicular Tg significantly suppressed the promoter activity, mRNA level, and protein level of SLC26A7 in rat thyroid FRTL-5 cells. In addition, follicular Tg inhibited the ability of TSH to induce the membrane localization of SLC26A7. In rat thyroid sections, the expression of SLC26A7 was weaker in follicles with a higher concentration of Tg, as evidenced by immunofluorescence staining. These results indicate that Tg stored in the follicular lumen is a feedback suppressor of the expression and membrane localization of SLC26A7, thereby downregulating the transport of iodide into the follicular lumen.

Genome-Wide Association Analysis and Genomic Prediction of Thyroglobulin Plasma Levels.

Thyroglobulin (Tg) is an iodoglycoprotein produced by thyroid follicular cells which acts as an essential substrate for thyroid hormone synthesis. To date, only one genome-wide association study (GWAS) of plasma Tg levels has been performed by our research group. Utilizing recent advancements in computation and modeling, we apply a Bayesian approach to the probabilistic inference of the genetic architecture of Tg. We fitted a Bayesian sparse linear mixed model (BSLMM) and a frequentist linear mixed model (LMM) of 7,289,083 variants in 1096 healthy European-ancestry participants of the Croatian Biobank. Meta-analysis with two independent cohorts (total n = 2109) identified 83 genome-wide significant single nucleotide polymorphisms (SNPs) within the ST6GAL1 gene (p<5×10-8). BSLMM revealed additional association signals on chromosomes 1, 8, 10, and 14. For ST6GAL1 and the newly uncovered genes, we provide physiological and pathophysiological explanations of how their expression could be associated with variations in plasma Tg levels. We found that the SNP-heritability of Tg is 17% and that 52% of this variation is due to a small number of 16 variants that have a major effect on Tg levels. Our results suggest that the genetic architecture of plasma Tg is not polygenic, but influenced by a few genes with major effects.

Targeted Next-Generation Sequencing of Congenital Hypothyroidism-Causative Genes Reveals Unexpected Thyroglobulin Gene Variants in Patients with Iodide Transport Defect.

Congenital iodide transport defect is an uncommon autosomal recessive disorder caused by loss-of-function variants in the sodium iodide symporter (NIS)-coding SLC5A5 gene and leading to dyshormonogenic congenital hypothyroidism. Here, we conducted a targeted next-generation sequencing assessment of congenital hypothyroidism-causative genes in a cohort of nine unrelated pediatric patients suspected of having a congenital iodide transport defect based on the absence of 99mTc-pertechnetate accumulation in a eutopic thyroid gland. Although, unexpectedly, we could not detect pathogenic SLC5A5 gene variants, we identified two novel compound heterozygous TG gene variants (p.Q29* and c.177-2A>C), three novel heterozygous TG gene variants (p.F1542Vfs*20, p.Y2563C, and p.S523P), and a novel heterozygous DUOX2 gene variant (p.E1496Dfs*51). Splicing minigene reporter-based in vitro assays revealed that the variant c.177-2A>C affected normal TG pre-mRNA splicing, leading to the frameshift variant p.T59Sfs*17. The frameshift TG variants p.T59Sfs*17 and p.F1542Vfs*20, but not the DUOX2 variant p.E1496Dfs*51, were predicted to undergo nonsense-mediated decay. Moreover, functional in vitro expression assays revealed that the variant p.Y2563C reduced the secretion of the TG protein. Our investigation revealed unexpected findings regarding the genetics of congenital iodide transport defects, supporting the existence of yet to be discovered mechanisms involved in thyroid hormonogenesis.

Thyrocyte cell survival and adaptation to chronic endoplasmic reticulum stress due to misfolded thyroglobulin.

The large secretory glycoprotein thyroglobulin is the primary translation product of thyroid follicular cells. This difficult-to-fold protein is susceptible to structural alterations that disable export of the misfolded thyroglobulin from the endoplasmic reticulum (ER), which is a known cause of congenital hypothyroidism characterized by severe chronic thyrocyte ER stress. Nevertheless, individuals with this disease commonly grow a goiter, indicating thyroid cell survival and adaptation. To model these processes, here we continuously exposed rat PCCL3 thyrocytes to tunicamycin, which causes a significant degree of ER stress that is specifically attributable to thyroglobulin misfolding. We found that, in response, PCCL3 cells down-regulate expression of the "tunicamycin transporter" (major facilitator superfamily domain containing-2A, Mfsd2a). Following CRISPR/Cas9-mediated Mfsd2a deletion, PCCL3 cells could no longer escape the chronic effects of high-dose tunicamycin, as demonstrated by persistent accumulation of unglycosylated thyroglobulin; nevertheless, these thyrocytes survived and grew. A proteomic analysis of these cells adapted to chronic ER protein misfolding revealed many hundreds of up-regulated proteins, indicating stimulation of ER chaperones, oxidoreductases, stress responses, and lipid biosynthesis pathways. Further, we noted increased phospho-AMP-kinase, suggesting up-regulated AMP-kinase activity, and decreased phospho-S6-kinase and protein translation, suggesting decreased mTOR activity. These changes are consistent with conserved cell survival/adaptation pathways. We also observed a less-differentiated thyrocyte phenotype with decreased PAX8, FOXE1, and TPO protein levels, along with decreased thyroglobulin mRNA levels. In summary, we have developed a model of thyrocyte survival and growth during chronic continuous ER stress that recapitulates features of congenital hypothyroid goiter caused by mutant thyroglobulin.

Thyroglobulin as a negative marker for malignancy in canine and human breast tumors.

Canine mammary gland tumors (CMTs) are the most common tumor type in female dogs. This study evaluated the expression pattern and role of thyroglobulin (Tg) in CMT and in human breast cancer (HBC). CMT samples were subjected to fine-needle aspiration, primary cell culture, and histopathology. The expression level of Tg was higher in benign CMT than in malignant CMT (mCMT) primary cells, particularly in the epithelial lineage. Moreover, treatment with Tg enhanced the sensitivity of doxorubicin in mCMT epithelial cells and mitigated proinflammatory response by increasing nuclear factor erythroid 2-related factor 2 (Nrf2). The proximal region of the Tg promoter was hypermethylated in mCMT epithelial cells, silencing Tg expression with concurrent downregulation of Nrf2-mediated antioxidant signaling. An identical pattern of Tg expression was observed in cytological and tissue samples. Tissue microarray analysis showed that Tg was highly expressed in normal and benign tissues when compared with their malignant counterparts, which was diminished along with higher histological grades. The survival rate was significantly higher in HBC patients with high Tg expression than those with low Tg expression. This study also showed that the progression of HBC is accompanied by the reduction of Tg expression along with augmentation of proinflammatory signaling. Our data suggested that Tg could be a negative indicator of malignancy in canine and human breast neoplasia.

Prediction of heparin binding of mutated short sequences of rat thyroglobulin.

BACKGROUND: Binding of thyroglobulin (Tg) to heparin is involved in Tg transcytosis via megalin. Rat Tg (rTg) binds to heparin through an exposed carboxyl terminal region (RELPSRRLKRPLPVK, Arg2489-Lys2503) rich in positively charged residues. This region is not entirely conserved in human Tg (hTg) (Arg2489-Glu2503, REPPARALKRSLWVE), resulting in lower affinity binding. Here, we developed a score to predict to what extent secondary structure modifications affect the heparin-binding ability of rTg. METHODS: We designed eight synthetic peptides, including one with the Arg2489-Lys2503 sequence of rTg (rTgP), one with the corresponding sequence of hTg (hTgP), and six "mutant" peptides, each carrying a point mutation obtained by replacing one amino acid residue of rTgP with the corresponding residue of hTgP. Heparin binding was assessed in solid-phase assays. The Bmax and the constants of dissociation (Kd) were calculated. RESULTS: Using a no-fee online service, we obtained predictions of peptide secondary structures and developed a scoring system to estimate to what extent mutations are expected to modify rTg secondary structure. The score was designated as Probability of Secondary Structure Change (PSSC) and it significantly correlated with the BMax (R = 0.942, P < 0.001) and the Kds (R = - 0.744, P < 0.01) of heparin binding of hTgP and of the "mutant" peptides. CONCLUSIONS: The PSSC score allows predicting to what extent point mutations are likely to affect the heparin-binding ability of short sequences of proteins: in this case rTg, regardless of whether mutations affect charge of the sequence. The secondary structure of Tg is likely to play a role in heparin binding.

Thyroid Gland Alterations in Old-Aged Wistar Rats: A Comprehensive Stereological, Ultrastructural, Hormonal, and Gene Expression Study.

The aim of the present study was to determine and elaborate on all changes in old-aged (OA) versus young-aged (YA) rat thyroids by using stereological, ultrastructural, hormonal, and gene expression analyses. We used 4- and 24-month-old male Wistar rats in our evaluation, presenting all changes in comparison with YA rats. Results showed that the thyroid parenchyma was characterized by higher absolute volumes of the gland, colloid, epithelium, and interstitium by 135, 135, 140, and 142% (p < 0.05) respectively, while the relative volumes of colloid and glands were unchanged. Ultrastructural analysis revealed less active glands, with smaller amounts of lysosomes, thyroglobulin (Tg) granules, and microvilli in the luminal colloid. Optical density values for thyroid peroxidase (TPO), Tg, and vascular-endothelial growth factor immunostaining remained unchanged; however, TPO and Tg exhibited visually stronger expression in small active follicles. Thyroxine (T4)-Tg, the relative intensity of fluorescence (RIF), serum T4, and the sodium-iodide symporter immunohistochemical and gene expressions decreased by 20, 40, 29, and 31% (p < 0.05), respectively, in OA thyroids. Pituitary thyroid-stimulating hormone (TSH) RIF increased by 44% (p < 0.05), but the TSH serum concentration remained unchanged. In conclusion, the obtained results indicate depression of the thyroid gland synthetic and secretory capacity with advanced age.

The decrease of T3 / T4 is not hypothyroidism - a new mutation of Serpina7 gene results in partial thyroglobulin deficiency.

To explore an unusual cause of the decrease of T3/T4 through a new mutation of TBG gene in a family, so as to avoid habitual thinking and reduce subsequent over treatment. TSH, free total T4, T3 and free T4, T3 were determined by automatic chemiluminescence immunoassay. The TBG mutation was identified by direct DNA sequencing. A frameshift mutation of p. l372ffs * 32 was found in the TBG gene (c.1114delc) of the patient by direct DNA sequencing, and the proband of the family was heterozygous. In vitro expression showed that the affinity of TBG for T4 decreased. Further examination of the family members showed that T3 and T4 were decreased, while FT3, FT4 and TSH were normal. If the patients with low TT4 and TT3 but normal TSH are found, the serum TBG level and related genes should be detected to determine whether it is TBG deficiency and avoid wrong treatment.

Newborn screening and single nucleotide variation profiling of TSHR, TPO, TG and DUOX2 candidate genes for congenital hypothyroidism.

High prevalence of congenital hypothyroidism (CH) among Indian newborns prompted us to establish population-specific reference ranges of TSH and to explore the contribution of the common genetic variants in TSHR, TPO, TG and DUOX2 genes towards CH. A total of 1144 newborns (593 males and 551 females) were screened for CH. SNV profiling (n = 22) spanning three candidate genes, i.e. TSHR, TPO and TG was carried out in confirmed CH cases (n = 45). In screen negative cases (n = 700), ten TSHR variants were explored to establish association with CH. No mutation found in DUOX2. The 2.5th to 97.5th percentiles of TSH in these newborns were 0.5 to 12.2 mU/L. In newborns with optimal birth weight, the cut-off was 10 mU/L. Lower or higher birth weight resulted in slightly higher TSH. Two TSHR variants, i.e. rs7144481 and rs17630128 were associated with agenesis, hypoplasia and goiter. The rs2268477 was associated with agenesis and hypoplasia. The rs1991517, rs2075176 and rs2241119 were associated with agenesis only. The rs7144481, rs17630128, rs1991517 and rs2268477 were associated with 2.17, 4.62, 2.91 and 2.29-fold increased risk for CH, respectively. Among the TPO variants, rs867983 and rs2175977 were associated with agenesis and goiter, respectively. Among the TG variants, rs2076740 showed association with agenesis and goiter. Two rare variants i.e. TPO g.IVS14-19 G>C and TG c.1262 C>T were observed in CH cases. No genetic variant identified in the two exons of DUOX2. To conclude, the current study established Indian population-specific normative values for TSH and demonstrates specific genotype-phenotype correlations among three candidate genes.

Morphological and functional changes in neonatally X-irradiated thyroid gland in rats.

Exposure to ionized radiation in childhood has been recognized as a risk factor for the development of thyroid cancer and possibly for other thyroid disorders. However, the effects of neonatal radiation exposure on thyroid morphology and functions have never been explored despite its potential importance. One-week-old male Wistar rats were subjected to cervical X-irradiation at 6 and 12 Gy. Animals were examined at the ages of 2, 8 and 18 weeks old. For comparison, 8-week-old rats were cervically X-irradiated at the same doses. Thyroid histology was examined by computer-assisted microscopy to measure areas of colloid and epithelium of thyroid follicles as well as epithelial heights. In rats that received cervical X-irradiation at 1 week old, the colloid size of thyroid follicles decreased at the age of 8 weeks old in a radiation-dose dependent manner. This morphological change was persistently found at 18 weeks old. There were no significant differences in serum total T3 or T4 levels among the groups. Serum TSH levels increased significantly in 8-week-old rats neonatally X-irradiated. Thyroglobulin (Tg) mRNA and protein expressions were significantly decreased in the neonatally-irradiated group while thyroid peroxidase mRNA express increased at 18 weeks old. None of these changes were observed in the rats X-irradiated at 8 weeks old. In conclusion, our results clearly demonstrated that neonatal rat thyroid was sensitive to ionized radiation, developing specific morphological changes characterized by smaller thyroid follicles along with changes in serum TSH levels and Tg expressions in the thyroid tissue.

Primary 3D Culture of Human Thyroid Follicle-Like Structures in Platelet Lysate-Based Gel.

The results of 3D culturing of human thyroid follicle-like structures in a gel based on platelet lysate at the gel-air interface are presented. During culturing up to 4 months, no new follicle-like structures were formed and none were destroyed. During the first 2 months, most follicle-like structures increased in size; then, their grown decelerated, but they retained viability. Ki-67+ cells were observed in the majority of follicle-like structures. Most of them produced thyroglobulin. Follicle-like structures get closer, the number of contacts between them increased, and cluster appeared. Thus, the developed 3D culturing system in a gel based on platelet lysate is an adequate approach for maintaining structure and functional activity of human follicle-like structures in vitro for at least 2 months.

Association between thyroglobulin polymorphisms and autoimmune thyroid disease: a systematic review and meta-analysis of case-control studies.

Emerging evidence revealed that thyroglobulin (TG) contributes to the development of autoimmune disease, and the relationship between TG and autoimmune thyroid disease (AITD) is still controversial. The aim of this study was to quantify the association between rs2076740, rs853326, rs180223, and rs2069550 TG polymorphisms and risk of AITD using a meta-analysis approach. We identified all studies that assessed the association between TG polymorphisms and AITD from PubMed, Embase, and Web of Science databases. A total of 3013 cases and 1812 controls from ten case-control studies were included. There was no significant associations found between rs2069550, rs180223, and rs853326 polymorphisms and AITD risk. The association between the rs2076740 polymorphism and AITD risk was significant in the codominant model (P = 0.005), suggesting the CC rs2076740 genotype might be a protective factor for AITD. Sensitivity analysis by removing one or two study changed the results in dominant rs2076740 and rs853326 and rs2069550 allele models (P = 0.016, 0.024, 0.027). Latitude and ethnicity significantly affected the association between rs2076740 and rs2069550 polymorphisms and AITD, indicating their protective effects in allele or dominant model (P = 0.012, 0.012, 0.012, 0.009, 0.009). The association between rs2076740, rs2069550, and rs853326 polymorphisms and AITD risk is significantly affected by study characteristics.

Relationship between the dimerization of thyroglobulin and its ability to form triiodothyronine.

Thyroglobulin (TG) is the most abundant thyroid gland protein, a dimeric iodoglycoprotein (660 kDa). TG serves as the protein precursor in the synthesis of thyroid hormones tetraiodothyronine (T4) and triiodothyronine (T3). The primary site for T3 synthesis in TG involves an iodotyrosine acceptor at the antepenultimate Tyr residue (at the extreme carboxyl terminus of the protein). The carboxyl-terminal region of TG comprises a cholinesterase-like (ChEL) domain followed by a short unique tail sequence. Despite many studies, the monoiodotyrosine donor residue needed for the coupling reaction to create T3 at this evolutionarily conserved site remains unidentified. In this report, we have utilized a novel, convenient immunoblotting assay to detect T3 formation after protein iodination in vitro, enabling the study of T3 formation in recombinant TG secreted from thyrocytes or heterologous cells. With this assay, we confirm the antepenultimate residue of TG as a major T3-forming site, but also demonstrate that the side chain of this residue intimately interacts with the same residue in the apposed monomer of the TG dimer. T3 formation in TG, or the isolated carboxyl-terminal region, is inhibited by mutation of this antepenultimate residue, but we describe the first substitution mutation that actually increases T3 hormonogenesis by engineering a novel cysteine, 10 residues upstream of the antepenultimate residue, allowing for covalent association of the unique tail sequences, and that helps to bring residues Tyr2744 from apposed monomers into closer proximity.

Genomic Profiling of Thyroid Cancer Reveals a Role for Thyroglobulin in Metastasis.

Papillary thyroid carcinoma (PTC) has a wide geographic variation in incidence; it is most common in Saudi Arabia, where it is only second to breast cancer as the most common cancer among females. Genomic profiling of PTC from Saudi Arabia has not been attempted previously. We performed whole-exome sequencing of 101 PTC samples and the corresponding genomic DNA to identify genes with recurrent somatic mutations, then sequenced these genes by using a next-generation gene-panel approach in an additional 785 samples. In addition to BRAF, N-RAS, and H-RAS, which have previously been shown to be recurrently mutated in PTC, our analysis highlights additional genes, including thyroglobulin (TG), which harbored somatic mutations in 3% of the entire cohort. Surprisingly, although TG mutations were not exclusive to mutations in the RAS-MAP kinase pathway, their presence was associated with a significantly worse clinical outcome, which suggests a pathogenic role beyond driving initial oncogenesis. Analysis of metastatic PTC tissue revealed significant enrichment for TG mutations (p < 0.001), including events of apparent clonal expansion. Our results suggest a previously unknown role of TG somatic mutations in the pathogenesis of PTC and its malignant evolution.