Falsely elevated thyroid hormone levels associated with fibrin interference in patients receiving oral anticoagulant therapy.

OBJECTIVE: Unique clinical courses were observed in two asymptomatic patients receiving warfarin who referred to our hospital because of suspected central hyperthyroidism. We eventually diagnosed these patients with falsely elevated thyroid hormone levels caused by macroscopically invisible fibrin. Although false results caused by fibrin interference in vitro have been identified in various immunoassays, especially in blood samples from patients receiving anticoagulant therapy, no studies on thyroid function testing have been reported. The experience in evaluating these cases prompted us to investigate the independent influence of oral anticoagulants via putative fibrin interference on thyroid function testing. METHODS: We retrospectively reviewed known contributing factors that affect thyroid function testing including age, gender, medication history, body mass index, estimated glomerular filtration rate, smoking status, alcohol consumption, and the seasons of hospital visits from participants who presented the Department of Health Checkup between April 2010 and December 2020. RESULTS: A propensity-matched analysis revealed that the median serum free thyroxine levels under oral anticoagulant were significantly higher (17.9 pmol/L, n = 60) than those without anticoagulants (16.0 pmol/L, n = 60; p < 0.001). It was noted that this difference was the largest among contributing factors we analyzed. No significant differences were noted in serum thyroid-stimulating hormone levels. CONCLUSIONS: We report two patients receiving warfarin with falsely elevated thyroid hormone levels caused by fibrin interference resembling central hyperthyroidism for the first time. Our retrospective study suggests that the medication status of oral anticoagulants should be considered when evaluating thyroid function tests.

Dose-Dependent Influence of Antithyroid Drugs on the Difference in Free Thyroxine Levels between Mothers with Graves' Hyperthyroidism and Their Neonates.

BACKGROUND: Several guidelines have recommended that the use of the lowest effective dose of antithyroid drugs (ATDs) that maintains maternal serum free thyroxine (FT4) levels at or moderately above the upper limit of the reference range is appropriate for fetal euthyroid status. However, little is known about whether ATD dosage affects the difference in serum FT4 levels between the mother and neonate. We conducted a retrospective study at a tertiary hospital in Japan to investigate the dose-dependent influence of ATDs on both maternal and fetal thyroid hormone status. MATERIALS AND METHODS: We retrospectively examined 62 pregnant women who delivered between 2007 and 2016 and were treated for Graves' hyperthyroidism with ATD at any stage during pregnancy. We selected individuals whose data on maternal FT4 level within 4 weeks of their deliveries and cord FT4 level of their infants at the time of delivery were available. Those with multiple pregnancies, iodine or glucocorticoid treatment, and fetal goiter detected by ultrasonography were excluded. RESULTS: After the exclusion criteria were applied, we recruited 40 individuals. The cord FT4 levels were significantly lower than the maternal FT4 levels in patients treated with high-dosage ATDs (methimazole >5 mg daily or propylthiouracil >100 mg daily). However, there were no significant differences between maternal and cord FT4 levels in patients treated with low-dosage ATDs (methimazole ≤5 mg daily or propylthiouracil ≤100 mg daily). We selected 35 individuals whose data on maternal thyrotropin receptor-binding inhibitory immunoglobulin (TBII) level were available. Multiple linear regression analysis adjusted for ATD dosage, maternal TBII level, and gestational period found that ATD dosage was a significant predictor of the difference in serum FT4 levels between the mother and neonate. In terms of maternal complications, multiple logistic regression analysis identified maternal free triiodothyronine (FT3) level as a significant predictor of the incidence of preterm delivery. CONCLUSIONS: We found a dose-dependent influence of ATDs on the difference in serum FT4 levels between mothers with Graves' hyperthyroidism and their neonates. Further studies to evaluate the optimal target FT4 and FT3 levels for the mother and neonate during pregnancy may improve the outcome of pregnant women with Graves' hyperthyroidism.

Functions of PIT1 in GATA2-dependent transactivation of the thyrotropin beta promoter.

Thyrotropin (TSH) is a heterodimer consisting of alpha and beta chains, and the beta chain (TSHbeta) is specific to TSH. The coexistence of two transcription factors, PIT1 and GATA2, is known to be essential for TSHbeta expression. Using kidney-derived CV1 cells, we investigated the role of PIT1 in the expression of Tshb gene. GATA2 Zn finger domain, which is known to recognize GATA-responsive elements (GATA-REs), is essential for cooperation by PIT1. Transactivation of TSHbeta promoter requires PIT1-binding site upstream to GATA-REs (PIT1-US), and the spacing between PIT1-US and GATA-REs strictly determines the cooperation between PIT1 and GATA2. Moreover, truncation of the sequence downstream to GATA-REs enabled GATA2 to transactivate the TSHbeta promoter without PIT1. The deleted region (nt -82/-52) designated as a suppressor region (SR) was considered to inhibit transactivation by GATA2. The cooperation of PIT1 with GATA2 was not conventional synergism but rather counteracted SR-induced suppression (derepression). The minimal sequence for SR was mapped to the 9 bp sequence downstream to GATA-REs. Electrophoretic mobility shift assay suggested that some nuclear factor exists in CV1 cells, which binds with SR and this interaction was blocked by recombinant PIT1. Our study indicates that major activator for the TSHbeta promoter is GATA2 and that PIT1 protects the function of GATA2 from the inhibition by SR-binding protein.

Essential role of GATA2 in the negative regulation of thyrotropin beta gene by thyroid hormone and its receptors.

Previously we reported that the negative regulation of the TSHbeta gene by T(3) and its receptor [thyroid hormone receptor (TR)] is observed in CV1 cells when GATA2 and Pit1 are introduced. Using this system, we further studied the mechanism of TSHbeta inhibition. The negative regulatory element (NRE), which had been reported to mediate T(3)-bound TR (T(3)-TR)-dependent inhibition, is dispensable, because deletion or mutation of NRE did not impair suppression. The reporter construct, TSHbeta-D4-chloramphenicol acetyltransferase, which possesses only the binding sites for Pit1 and GATA2, was activated by GATA2 alone, and this transactivation was specifically inhibited by T(3)-TR. The Zn finger region of GATA2 interacts with the DNA-binding domain of TR in a T(3)-independent manner. The suppression by T(3)-TR was impaired by overexpression of a dominant-negative type TR-associated protein (TRAP) 220, an N- and C-terminal deletion construct, indicating the participation of TRAP220. Chromatin immunoprecipitation assays with a thyrotroph cell line, TalphaT1, revealed that T(3) treatment recruited histone deacetylase 3, reduced the acetylation of histone H4, and caused the dissociation of TRAP220 within 15-30 min. The reduction of histone H4 acetylation was transient, whereas the dissociation of TRAP220 persisted for a longer period. In the negative regulation of the TSHbeta gene by T(3)-TR we report that 1) GATA2 is the major transcriptional activator of the TSHbeta gene, 2) the putative NRE previously reported is not required, 3) TR-DNA-binding domain directly interacts with the Zn finger region of GATA2, and 4) histone deacetylation and TRAP220 dissociation are important.

Thyroid-hormone-dependent negative regulation of thyrotropin beta gene by thyroid hormone receptors: study with a new experimental system using CV1 cells.

The molecular mechanism involved in the liganded thyroid hormone receptor suppression of the TSHbeta (thyroid-stimulating hormone beta, or thyrotropin beta) gene transcription is undetermined. One of the main reasons is the limitation of useful cell lines for the experiments. We have developed an assay system using non-pituitary CV1 cells and studied the negative regulation of the TSHbeta gene. In CV1 cells, the TSHbeta-CAT (chloramphenicol acetyltransferase) reporter was stimulated by Pit1 and GATA2 and suppressed by T3 (3,3',5-tri-iodothyronine)-bound thyroid hormone receptor. The suppression was dependent on the amounts of T3 and the receptor. Unliganded receptor did not stimulate TSHbeta activity, suggesting that the receptor itself is not an activator. Analyses using various receptor mutants revealed that the intact DNA-binding domain is crucial to the TSHbeta gene suppression. Co-activators and co-repressors are not necessarily essential, but are required for the full suppression of the TSHbeta gene. Among the three receptor isoforms, beta2 exhibited the strongest inhibition and its protein level was the most predominant in a thyrotroph cell line, TalphaT1, in Western blotting. The dominant-negative effects of various receptor mutants measured on the TSHbeta-CAT reporter were not simple mirror images of those in the positive regulation under physiological T3 concentration.

Beta-catenin/Tcf-1-mediated transactivation of cyclin D1 promoter is negatively regulated by thyroid hormone.

Cyclin D1 is an oncogenic cyclin frequently over-expressed in cancer. To examine the effect of thyroid hormone (T3) and its receptor (TR) on the transcription of cyclin D1 gene, we co-transfected the chloramphenicol acetyl-transferase (CAT) reporter plasmid containing cyclin D1 promoter together with the expression plasmids for TRbeta1 and wild-type or mutant beta-catenin (SA) into 293T cells. In the presence of T3, beta-catenin-dependent transactivation of cyclin D1 promoter was suppressed by co-transfection of TRbeta1. The suppression by T3/TRbeta1 was in a dose-dependent manner. The CAT reporter gene in which Tcf/Lef-1 sites were fused to heterologous promoter was also suppressed by T3/TRbeta1. Furthermore, inhibition of endogenous wild-type beta-catenin by T3/TRbeta1 was observed in SW480 colon carcinoma cells with mutation of the adenomatous polyposis coli gene. These results indicate that the T3-bound TR inhibits the transcription of cyclin D1 through the Tcf/Lef-1 site, which is positively regulated by the Wnt-signaling pathway.