Thyroglobulin Gene Research Articles

ANALYSIS OF THE GENETIC STRUCTURE OF THE POLISH BEEF BREED BY MOLECULAR MARKERS OF MEAT QUALITY INDICATORS PRODUCTIVITY

The results of determining the genotypes of Polish beef breed animals based on polymorphism of the thyroglobulin (TG 5) and calpain (CAPN1 530) genes are given. The PCR-PDRF method was used for the analysis. Based on the obtained data, the specifics of the genetic structure of the studied population were established based on the distribution of frequencies of alleles and genotypes. For the thyroglobulin gene, animals with the homozygous SS genotype had a quantitative advantage, which led to a high concentration of the C allele (0.775). For the calpain gene, a high frequency of the desired G allele was found in the studied group of animals (0.539). Based on the results of the analysis, the prospects of the researched group of animals of the Polish meat breed were determined for establishing an associative relationship of various allelic variants of the thyroglobulin and calpain genes with the parameters of the quality characteristics of the final product – marbling and tenderness of meat.

Effects of GH L127V and TG5 C422T polymorphisms on the hormonal profile, slaughter traits, and meat quality of Hereford bulls.

The creation of objective methods for the evaluation and improvement of quantitative and qualitative indicators of meat productivity in farm animals should be based on a comprehensive analysis of the genetic, physiological, and biochemical parameters of the animal. This study aimed to investigate the effects of growth hormone (GH) and thyroglobulin (TG5) gene polymorphisms on the hormonal status, slaughter traits, and chemical, amino acid, and fatty acid composition of meat in Hereford bulls. Hereford bulls (n = 9) were reared under the same feeding and housing conditions until the age of 21 months, after which they were slaughtered. Polymerase chain reaction-restriction fragment length polymorphism was performed for genotyping GH L127V and TG5 C422T polymorphisms. The experimental animals were evaluated to determine slaughter traits (including pre-slaughter weight, carcass, and internal fat weight and yield), chemical, fatty acid, and amino acid composition of ground beef, and hormonal status using serum concentrations of GH, triiodothyronine, and thyroxine. Animals with the valine homozygous (VV) genotype of GH had the maximum serum GH level of 9.33 mIU/mL (p = 0.10) higher than leucine homozygous (LL) genotype carriers. Individuals with the LL genotype outperformed V-allele carriers in serum thyroxine (T4) concentration by 21.3-30.5 nmol/L (16.15%-24.86%; p < 0.01-0.05). Genetic differentiation induced by TG5 C422T polymorphism was determined to a lesser extent by the hormonal status of the Hereford animals. The V-allele was associated with increased carcass weight, with VV homozygotes significantly outperforming LL individuals by 45.0 kg (13.61%; p < 0.05). The T allele at the TG5 gene polymorphism was associated with more intense lipogenesis and less protein synthesis in muscle tissue and these effects were enhanced in the homozygous state. Young animals with the TT variant of the TG5 gene exhibited a significantly superior polyunsaturated fatty acid/saturated fatty acid ratio of 0.012 units (p < 0.01). Carriers of the LL genotype were characterized by minimum amino acid content in muscle tissue. Heterozygous bulls exceeded LL homozygotes in the sum of essential amino acids by 3.09% (p = 0.10) and non-essential amino acids by 1.9% (p < 0.05). The development of breeding programs for the Hereford breed should be carried out considering genetic features that determine the formation of economic traits in animals. Analysis of polymorphisms in the TG5 gene is a promising method for the early diagnosis of the fatty acid composition of beef. Identification of polymorphisms in the GH gene allows the prediction of higher productivity potential and amino acid composition of meat. The different effects of the GH and TG5 genes on the development of various economic traits allowed us to determine further vectors for scientific research on their complex associations in Hereford cattle, which will be useful for planning effective breeding schemes.

Clinical and molecular study of patients with thyroid dyshormogenesis and variants in the thyroglobulin gene.

Defects in any thyroid hormone synthesis steps cause thyroid dyshormonogenesis (THD). THD due to thyroglobulin (TG) gene variants is a cause of congenital hypothyroidism (CH) with a wide clinical spectrum, ranging from mild to severe permanent hypothyroidism. We present high-throughput sequencing results of patients with TG variants. A CH high-throughput sequencing-panel of the main genes involved in the regulation of thyroid hormonogenesis was performed to identify those TG variants that may be related to patient THD phenotype. We identified 21 TG gene variants in 19 patients (11.8%) which could explain their phenotype. Ten of those (47.6%) were not previously described. CH was biochemically severe in these 19 patients. Eight of them were reevaluated after one month of discontinuing LT4 treatment and all had severe permanent hypothyroidism. We also identified another 16 patients who presented heterozygous TG variants, of whom, at reevaluation, five had mild permanent and only one had severe permanent hypothyroidisms. In this study, 10 novel and 11 previously reported variants in the TG gene have been identified that could explain the phenotype of 19 patients from non-consanguineous families from a large THD cohort. Although not all these TG gene variants can explain all the patients' THD phenotypes, some of them had severe or mild permanent hypothyroidism at reevaluation.

Developmental toxicity and mechanism of dibutyl phthalate and alternative diisobutyl phthalate in the early life stages of zebrafish (Danio rerio)

Diisobutyl phthalate (DiBP), is widely chemical replacement for Dibutyl phthalate (DBP). Although DBP and DiBP have been detected in surface water worldwide, few studies to date have systematically assessed the risks of DBP and its alternatives to aquatic organisms. The present study compared DBP and DiBP for their individual and joint toxicity as well as thyroid hormone levels in zebrafish embryo. Transcripts of key genes related to the hypothalamic-pituitary-thyroid (HPT) axis were investigated in developing zebrafish larvae by application of real time polymerase chain reaction. The median half-lethal concentrations of DBP and DiBP to zebrafish at 96 h were 0.545 mg L−1 and 1.149 mg L−1, respectively. The joint toxic effect of DBP–DiBP (0.25–0.53 mg L−1) with the same ratio showed a synergistic effect. Thyroid hormones levels increased with exposure to 10 μg L−1 of DBP or 50 μg L−1 of DiBP, and exposure to both compounds significantly increased thyroid gland-specific transcription of thyroglobulin gene (tg), hyronine deiodinase (dio2), and transthyretin (ttr), indicating an adverse effect associated with the HPT axis. Molecular docking results indicated that DBP (−7.10 kcal/M and −7.53 kcal/M) and DiBP (−6.63 kcal/M and −7.42 kcal/M) had the same docking energy with thyroid hormone receptors. Our data facilities an understand of potential harmful effects of DBP and its alternative (DiBP).

Use of molecular marker to evaluate marbling in Senepol cattle

Marbling, one of the main characteristics related to beef quality, has led to the development of numerous techniques for its measurement, such as the use of carcass ultrasound, identifying breeds and/or strains that present greater marbling without the need to slaughter the animals and the use of molecular markers, which confer the genes alleles that may have a negative or positive effect for a given trait. Currently, Thyroglobulin (TG) has been studied as a gene related to marbling in bovine species, therefore, the objective of this work was to verify the expression of polymorphisms of TG gene in 62 Senepol steers, using the PCR-RFLP technique. For that, samples of broom hairs from the tail of the animals were collected for the extraction of genomic DNA. The pair of primers used in the PCR reactions were designed according to GenBank and the restriction enzyme chosen was the XmnI endonuclease. To visualize the results, electrophoresis in a 2% agarose gel was performed. Of the 62 animals evaluated, 49 were positive for Thyroglobulin gene expression, suggesting the veracity of the association of this gene with marbling, which may allow the selection of Senepol animals with greater aptitude for marbling.

Cloning of recombinant thyroglobulin

The aim of the study is to clone cDNA fragments of mRNA of the thyroglobulin gene into а vector and introduce it into prokaryotic cell lines for possible use in the diagnosis of autoimmune and oncological diseases of the human thyroid gland. Thyroid hormones are synthesized by thyrocytes of mammalian thyroid tissue from the large protein thyroglobulin having a molecular weight of 330 kDa. The thyroid hormones thyroxine and triiodothyronine are small molecules that have large biological effects. They regulate metabolism in almost all cells, and are important for the development of the central nervous system, musculoskeletal system and lungs. These are also the only hormones that contain iodine and are synthesized partly inside and partly outside of cells. A dimeric glycoprotein is called thyroglobulin.

Research of thyroglobulin gene polymorphism in cows of Kostroma and black-and-white breeds of Kostroma region

Relevance. The genotype of the animal plays a significant role in realizing its productive potential, along with other factors affecting dairy productivity. Marker-oriented breeding (MAS) of cattle, which utilizes genetic markers associated with economically valuable traits, has gained popularity in the last two decades. One such marker is the thyroglobulin (TG) gene, which is involved in the production of a thyroid prohormone.Methods.The research was conducted on cows of Kostroma and black-and-white breeds at breeding farms in the Kostroma region. Genetic material was obtained from the tail vein of the cows using individual vacuum systems with an anticoagulant. The polymorphism of the TG gene (rs135751032) was determined using RT-PCR and detection by hybridization-fluorescence method. The results were processed using biometric analysis and calculation of population-genetic indicators.Results. In the populations of cows of the Kostroma and black-and-white breeds of the Kostroma region, the predominance of carriers of the CC genotype and the C allele of the thyroglobulin gene was established. At the same time, among cows of these breeds, there is a tendency to higher milk productivity in heterozygous animals. The nature of the associative relationship between TG genotypes and milk productivity indicators differed significantly for each individual herd. This indicates the need to study TG in combination with other DNA markers of milk productivity and metabolism. Statistically significant differences between cows of different TG genotypes were found only in full-aged animals, which perhaps indicates the prospects of using TG to determine the genetic potential of productive longevity of cows.

Association study between SNP markers located in meat quality candidate genes with intramuscular fat content in an endangered dual-purpose cattle population.

The aim of this study was to associate single nucleotide polymorphisms (SNP) of the bovine calcium-activated neutral protease µ-calpain, calpastatin, diacylglycerol-O-acyltransferase, adipose fatty acid binding protein, retinoic acid receptor-related orphan receptor C (RORC), and thyroglobulin (TG) gene with intramuscular fat content (IMF). Therefore, 542 animals of the cattle breed "Rotes Höhenvieh" (RHV) were phenotyped for IMF. Genotyping of the animals was performed using polymerase chain reaction-restriction fragment length polymorphism tests for six SNP from candidate genes for meat quality traits. In addition, we calculated allele substitution and dominance effects on IMF. A subgroup of animals (n = 44, reduced dataset) with extraordinary high IMF was analyzed separately. The mean IMF content was 2.5% (SD: 2.8) but ranged from 0.02% to 23.9%, underlining the breeds' potential for quality meat production. Allele and genotype frequencies for all SNP were similar in the complete and reduced dataset. Association analyses in the complete dataset revealed the strongest effects of RORC on IMF (P = 0.075). The log-transformed least-squares mean for IMF of genotype g.3290GG was 0.45 ± 0.16, 0.26 ± 0.14 for genotype g.3290GT, and 0.32 ± 0.14 for genotype g.3290TT. In the reduced dataset, we found a significant effect (P < 0.05) of the g.422C>T-SNP of TG on IMF, with highest IMF for genotype CT (0.91 ± 0.17), lowest IMF for genotype TT (0.37 ± 0.25), and medium IMF for genotype CC (0.59 ± 0.16; log-transformed values). Compared to the complete dataset, allele substitution effects increased in the reduced dataset for most of the SNP, possibly due to the selective genotyping strategy, with focus on animals with highest IMF implying strong phenotypic IMF contrast. Dominance effects were small in both datasets, related to the high heritability of IMF. Results indicated RHV breed particularities regarding the effects of meat quality genes on IMF. An explanation might be the breeding history of RHV with focus on adaptation and resilience in harsh outdoor systems. Consequently, it is imperative to develop breed-specific selection strategies. Allele substitution and dominance effects were in a similar direction in both datasets, suggesting the same breeding approaches for different RHV strains in different regions. Nevertheless, a selective genotyping approach (reduced dataset), contributed to more pronounced genotype effect differences on IMF and dominance values.

A Novel Transgenic Model to Study Thyroid Axis Activity in Early Life Stage Medaka.

Identifying endocrine disrupting chemicals in order to limit their usage is a priority and required according to the European Regulation. There are no Organization for Economic Co-operation and Development (OECD) test guidelines based on fish available for the detection of Thyroid axis Active Chemicals (TACs). This study aimed to fill this gap by developing an assay at eleuthero-embryonic life stages in a novel medaka (Oryzias latipes) transgenic line. This transgenic line expresses green fluorescent protein (GFP) in thyrocytes, under the control of the medaka thyroglobulin gene promoter. The fluorescence expressed in the thyrocytes is inversely proportional to the thyroid axis activity. When exposed for 72 h to activators (triiodothyronine (T3) and thyroxine (T4)) or inhibitors (6-N-propylthiouracil (PTU), Tetrabromobisphenol A (TBBPA)) of the thyroid axis, the thyrocytes can change their size and express lower or higher levels of fluorescence, respectively. This reflects the regulation of thyroglobulin by the negative feedback loop of the Hypothalamic-Pituitary-Thyroid axis. T3, T4, PTU, and TBBPA induced fluorescence changes with the lowest observable effect concentrations (LOECs) of 5 μg/L, 1 μg/L, 8 mg/L, and 5 mg/L, respectively. This promising tool could be used as a rapid screening assay and also to help decipher the mechanisms by which TACs can disrupt the thyroid axis in medaka.

The Relationship Between the E12 Polymorphism of The Thyroglobulin Gene and Hyperthyroidism Diseases in Affected People in Samarra City

The Relationship Between the E12 Polymorphism of The Thyroglobulin Gene and Hyperthyroidism Diseases in Affected People in Samarra City

The highly and perpetually upregulated thyroglobulin gene is a hallmark of functional thyrocytes.

Abnormalities are indispensable for studying normal biological processes and mechanisms. In the present work, we draw attention to the remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene (Tg). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so-called transcription loops, easily observable by light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both alleles remain highly active over the entire life of the cell, i.e., for months or years depending on the species. We demonstrate that this high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We provide evidence that Tg is not influenced by the thyroid hormone status, does not oscillate round the clock and is expressed during both the exocrine and endocrine phases of thyrocyte activity. We conclude that the thyroglobulin gene represents a unique and valuable model to study the maintenance of a high transcriptional upregulation.

Peculiarities of circadian regulation of the thyroid gland

Circadian rhythmicity is an autonomous period of organ cells’ functioning that is controlled by feedback mechanisms and specific genes. The thyroid gland, as a peripheral organ of the endocrine system, is subordinate to the adenohypophysis and pineal gland. Melatonin, as one of the main mediators of circadian influence, is well known for its antitumor and regulatory effects on a number of tissues, but its relationship with the hormone-producing activity of the thyroid gland has not been sufficiently studied. The purpose of the work was to analyze the current data on circadian regulation of the thyroid gland by examining evidence-based scientific publications from open databases over the past five years. Melatonin-producing pineal cells possess the homeotic genes Otx2 and Crx whose deactivation leads to a significant decrease in melatonin production by the pineal gland. In addition to pineal cells, melatonin can be synthesized by adenohypophysis-independent parafollicular cells. The effect of melatonin on thyrocytes is provided by binding to MT1 receptors and affecting the expression of thyroglobulin, RX-8, and TTF-1 (NKX2-1) genes. Under conditions of short photoperiod, melatonin inhibits the production of thyroid-stimulating hormone (TSH) β, which, in turn, acts on hypothalamic tanycytes by regulating the balance of deiodinase (Dio2/Dio3). This leads to a well-regulated seasonal control of the release of the thyroid hormone triiodothyronine. Conclusions. Circadian regulation of thyrocyte activity occurs under the influence of melatonin, which is secreted by pinealocytes and sometimes by parafollicular cells. TSH is secreted with a certain circadian periodicity under the influence of the activation of chronorhythmic genes. Hormone replacement therapy should be aimed not only at restoring the levels of pituitary hormones, but also at restoring the biological rhythm of TSH secretion. Melatonin affects the circadian work of thyrocytes, which is proven by determining the expression of the chronorhythm genes Bmal-1, Dio2, TTF-1. In thyroid pathology, there is a violation of circadian rhythms, a decrease in melatonin levels, and a decrease in the expression of Clock genes.

Effects of leptin and thyroglobulin gene polymorphisms on beef colour in Holstein bulls for slaughter in Turkey

n the present work, live weight (LW), hot carcass weight (HCW), and beef colour values of Turkish Holstein bull (THBs) samples, and their relationship with single nucleotide polymorphisms (SNPs) variants were determined. E2JW and E2FB variants of leptin (LEP), and C422T variant of thyroglobulin (TG) genes were determined in 100 heads of THBs by polymerase chain reaction fragment length polymorphism (PCR-RFLP). Genotyping was carried out by capillary electrophoresis. The colour of raw and cooked beefs was spectrophotometrically measured before and after cooking. The cooked beefs were significantly brighter in the LEP E2JW AA and AT variants than in the TT genotype (p &lt; 0.05). Based on b* of raw beefs, the yellowish colour density in the LEP E2JW AA genotype variant was significantly higher than in AT and TT (p &lt; 0.05). The most significant correlation was determined between b* and L* (0.695), and b* and a* (0.694) of raw beefs, while the correlation coefficient between LW and HCW was found to be 0.604 (p &lt; 0.01). The LEP E2JW AA marker genotype for cattle with brighter and more intense beef, and the LEP E2JW TT variant genotype to increase beef yield should be selected as a study by using MAS method at an early age. Also, AT / CT / CC marker genotypes of THBs should be selected in LEP E2JW / E2FB / TG C422T marker loci, respectively to generate more income from the increase in LW and HCW.

The p.Pro2232Leu variant in the ChEL domain of thyroglobulin gene causes intracellular transport disorder and congenital hypothyroidism.

Thyroglobulin (TG), the predominant glycoprotein of the thyroid gland, functions as matrix protein in thyroid hormonegenesis. TG deficiency results in thyroid dyshormonogenesis. These variants produce a heterogeneous spectrum of congenital goitre, with an autosomal recessive mode of inheritance. The purpose of this study was to identify and functionally characterize new variants in the TG gene in order to increase the understanding of the molecular mechanisms responsible for thyroid dyshormonogenesis. A total of four patients from two non-consanguineous families with marked alteration of TG synthesis were studied. The two families were previously analysed in our laboratory, only one deleterious allele, in each one, was detected after sequencing the TG gene (c.2359 C > T [p.Arg787*], c.5560 G > T [p.Glu1854*]). These findings were confirmed in the present studies by Next-Generation Sequencing. The single nucleotide coding variants of the TG gene were then analyzed to predict the possible variant causing the disease. The p.Pro2232Leu (c.6695 C > T), identified in both families, showing a low frequency population in gnomAD v2.1.1 database and protein homology, amino acid prediction, and 3D modeling analysis predict a potential pathogenic effect of this variant. We also transiently express p.Pro2232Leu in a full-length rat TG cDNA clone and confirmed that this point variant was sufficient to cause intracellular retention of mutant TG in HEK293T cells. Consequently, each family carried a compound heterozygous for p.Arg787*/p.Pro2232Leu or p.Glu1854*/p.Pro2232Leu variants. In conclusion, our results confirm the pathophysiological importance of altered TG folding as a consequence of missense variants located in the ChEL domain of TG.

Utility of Antibodies in the Diagnoses of Thyroid Diseases: A Review Article.

Thyroid problems are among the most widespread endocrine illnesses, affectingindividuals in India andthe global population. A thyroid function test is used to diagnose, screen, and monitor patients. Hyperthyroidism is a clinical condition due to excessive circulation of thyroid hormone; in contrast, hypothyroidism is due to a deficiency of thyroid hormone. Graves' disease (GD) is a form of hyperthyroidism due to thyroid-stimulating hormonereceptor autoantibodies (TRAb), and anti-thyroid peroxidase antibodies (anti-TPO antibodies). The most common reason for hypothyroidism is Hashimoto's thyroiditis (HT), in which patients have thyroid receptor antibodies (TRAb), antibodies to thyroid peroxidase (TPO), and thyroglobulin antibodies.Many essential genes, including the thyroid-specific genes thyroglobulin (TSGT), TSH-receptor gene, human leukocyte antigen (HLA) genes, cytotoxic T lymphocyte-associated antigen (CTLA)genes, thyroglobulin gene, vitamin D receptor gene, and many immune-regulatory geneswere associated with autoimmune thyroid diseases' (AITDs') etiology. This review paper aims to determine if antibodies are beneficial in detecting autoimmune thyroid disease or not. We have also discussed the etiology of autoimmune thyroid illness, serum antibodies in autoimmune thyroid disease, pathophysiology, and TSH receptor features.

Connection between gene markers with milk production traits of Ukrainian dairy cows

The purpose of this study was to obtain information on the genotyping of cows of Ukrainian dairy breeds for the presented loci capa-casein gene (CSN3), thyroglobulin (TG-5) gene, leptin (LEP), pituitary-specific transcription factor (Pit-1), and beta-lactoglobulin (BLG) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and to test their association with milk production. The influence of the genotype on milk yield, fat and protein content in the milk of cows of Ukrainian selection has been established. The highest levels of milk productivity traits were expected from animals with the CT genotype for the LEP gene, AA for the CSN3 gene, CC for the TG5 gene, and AA and BB for the Pit-1 gene. But at the same time, milk obtained from cows with CSN3 BB genotype were characterized by the best cheese suitability. The effect of the homo- or heterozygous state of BLG on the signs of milk production was less noticeable, and the homozygous TT genotype for the TG5 gene was not found in any of the studied breeds. The results obtained can be used in the practical work of breeding farms along with traditional methods of selection, control and preservation of the genetic diversity of specific herds at an optimal level.

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.

Thyroid Dysfunction of Zebrafish (Danio rerio) after Early-Life Exposure and Discontinued Exposure to Tetrabromobiphenyl (BB-80) and OH-BB-80.

3,3',5,5'-Tetrabromobiphenyl (BB-80) was once used as additive flame retardants. Whether its early exposure and discontinued exposure alter thyroid function remains unknown. We investigate adverse effects after early-life exposure and discontinued exposure to BB-80 and hydroxylated BB-80 (OH-BB-80) on thyroid hormone (TH) levels, thyroid tissue, and transcriptome profiles in zebrafish larvae. BB-80 at 10 μg/L induces pathological changes of thyroid with reduced thyroid follicles in larvae (P < 0.05), whereas OH-BB-80 significantly increases T4 and T3 contents (1.8 and 2.5 times of the control, P < 0.05) at 14 days postfertilization (dpf) without morphological thyroid alterations. BB-80 and OH-BB-80 cause transcriptome aberrations with key differentially expressed genes involved in the disruption of TH synthesis and signal transduction (BB-80 at 14 dpf) or TH pathway activation (OH-BB-80 at 21 dpf). After 7 days of discontinued exposure, thyroglobulin (tg) and thyroid peroxidase (tpo) genes are downregulated (P < 0.05) by 52 and 48% for BB-80 and by 49 and 39% for OH-BB-80, respectively; however, the whole-body TH levels fail to fully recover, and the locomotor activity is impaired more by BB-80. Our results indicate significant adverse impacts of BB-80 and OH-BB-80 on TH homeostasis and thyroid function of zebrafish.

TSH stimulation of human thyroglobulin and thyroid peroxidase gene transcription is partially dependent on internalization

The TSH receptor (TSHR) is the major regulator of thyroid hormone biosynthesis in human thyrocytes by regulating the transcription of a number of genes including thyroglobulin (TG) and thyroperoxidase (TPO). Until recently, it was thought that TSHR initiated signal transduction pathways only at the cell-surface and that internalization was primarily involved in TSHR desensitization and downregulation. Studies primarily in mouse cells showed that TSHR internalization regulates gene transcription at an intracellular site also. However, this has not been shown for genes involved in thyroid hormone biosynthesis in human thyrocytes. We used human thyrocytes in primary culture. In these cells, the dose-response to TSH for gene expression is biphasic with low doses upregulating gene expression and higher doses decreasing gene expression. We used two approaches to inhibit internalization. In the first, we used inhibitors of dynamins, dynasore and dyngo-4a. Pretreatment with dynasore or dyngo-4a markedly inhibited TSH upregulation of TG and TPO mRNAs, as well as TG secretion. In the second, we used knockdown of dynamin 2, which is the most abundant dynamin in human thyrocytes. We showed that dynamin 2 knockdown inhibited TSHR internalization and decreased the TSH-stimulated levels of TG and TPO mRNAs and proteins. Lastly, we showed that the level of the activatory transcription factor phosphorylated cAMP response element binding protein (pCREB) in the cell nuclei was reduced by 68% when internalization was inhibited. We conclude that upregulation of genes involved in thyroid hormone synthesis in human thyrocytes is, in part, dependent on internalization leading to nuclear localization of an activated transcription factor(s).

Molecular insights into the role of genetic determinants of congenital hypothyroidism.

In our previous studies, we have demonstrated the association of certain variants of the thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase (TPO), and thyroglobulin (TG) genes with congenital hypothyroidism. Herein, we explored the mechanistic basis for this association using different in silico tools. The mRNA 3'-untranslated region (3'-UTR) plays key roles in gene expression at the post-transcriptional level. In TSHR variants (rs2268477, rs7144481, and rs17630128), the binding affinity of microRNAs (miRs) (hsa-miR-154-5p, hsa-miR-376a-2-5p, hsa-miR-3935, hsa-miR-4280, and hsa-miR-6858-3p) to the 3'-UTR is disrupted, affecting post-transcriptional gene regulation. TPO and TG are the two key proteins necessary for the biosynthesis of thyroid hormones in the presence of iodide and H2O2. Reduced stability of these proteins leads to aberrant biosynthesis of thyroid hormones. Compared to the wild-type TPO protein, the p.S398T variant was found to exhibit less stability and significant rearrangements of intra-atomic bonds affecting the stoichiometry and substrate binding (binding energies, ΔG of wild-type vs. mutant: ‒15 vs. ‒13.8 kcal/mol; and dissociation constant, Kd of wild-type vs. mutant: 7.2E-12 vs. 7.0E-11 M). The missense mutations p.G653D and p.R1999W on the TG protein showed altered ΔG (0.24 kcal/mol and 0.79 kcal/mol, respectively). In conclusion, an in silico analysis of TSHR genetic variants in the 3'-UTR showed that they alter the binding affinities of different miRs. The TPO protein structure and mutant protein complex (p.S398T) are less stable, with potentially deleterious effects. A structural and energy analysis showed that TG mutations (p.G653D and p.R1999W) reduce the stability of the TG protein and affect its structure-functional relationship.