Triiodothyronine regulates angiogenic growth factor and cytokine secretion by isolated human decidual cells in a cell-type specific and gestational age-dependent manner.

STUDY QUESTION: Does triiodothyronine (T3) regulate the secretion of angiogenic growth factors and cytokines by human decidual cells isolated from early pregnancy? SUMMARY ANSWER: T3 modulates the secretion of specific angiogenic growth factors and cytokines, with different regulatory patterns observed amongst various isolated subpopulations of human decidual cells and with a distinct change between the first and second trimesters of pregnancy. WHAT IS KNOWN ALREADY: Maternal thyroid dysfunction during early pregnancy is associated with complications of malplacentation including miscarriage and pre-eclampsia. T3 regulates the proliferation and apoptosis of fetal-derived trophoblasts, as well as promotes the invasive capability of extravillous trophoblasts (EVT). We hypothesize that T3 may also have a direct impact on human maternal-derived decidual cells, which are known to exert paracrine regulation upon trophoblast behaviour and vascular development at the uteroplacental interface. STUDY DESIGN, SIZE, DURATION: This laboratory-based study used human decidua from first (8-11 weeks; n = 18) and second (12-16 weeks; n = 12) trimester surgical terminations of apparently uncomplicated pregnancies. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary cultures of total decidual cells, and immunomagnetic bead-isolated populations of stromal-enriched (CD10+) and stromal-depleted (CD10-) cells, uterine natural killer cells (uNK cells; CD56+) and macrophages (CD14+) were assessed for thyroid hormone receptors and transporters by immunocytochemistry. Each cell population was treated with T3 (0, 1, 10, 100 nM) and assessments were made of cell viability (MTT assay) and angiogenic growth factor and cytokine secretion (immunomediated assay). The effect of decidual cell-conditioned media on EVT invasion through Matrigel(®) was evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: Immunocytochemistry showed the expression of thyroid hormone transporters (MCT8, MCT10) and receptors (TRα1, TRß1) required for thyroid hormone-responsiveness in uNK cells and macrophages from the first trimester. The viability of total decidual cells and the different cell isolates were unaffected by T3 so changes in cell numbers could not account for any observed effects. In the first trimester, T3 decreased VEGF-A secretion by total decidual cells (P < 0.05) and increased angiopoietin-2 secretion by stromal-depleted cells (P < 0.05) but in the second trimester total decidual cells showed only increased angiogenin secretion (P < 0.05). In the first trimester, T3 reduced IL-10 secretion by total decidual cells (P < 0.05), and reduced granulocyte macrophage colony stimulating factor (P < 0.01), IL-8 (P < 0.05), IL-10 (P < 0.01), IL-1ß (P < 0.05) and monocyte chemotactic protein -1 (P < 0.001) secretion by macrophages, but increased tumour necrosis factor-α secretion by stromal-depleted cells (P < 0.05) and increased IL-6 by uNK cells (P < 0.05). In contrast, in the second trimester T3 increased IL-10 secretion by total decidual cells (P < 0.01) but did not affect cytokine secretion by uNK cells and macrophages. Conditioned media from first trimester T3-treated total decidual cells and macrophages did not alter EVT invasion compared with untreated controls. Thus, treatment of decidual cells with T3 resulted in changes in both angiogenic growth factor and cytokine secretion in a cell type-specific and gestational age-dependent manner, with first trimester decidual macrophages being the most responsive to T3 treatment, but these changes in decidual cell secretome did not affect EVT invasion in vitro. LIMITATIONS, REASONS FOR CAUTION: Our results are based on in vitro findings and we cannot be certain if a similar response occurs in human pregnancy in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Optimal maternal thyroid hormone concentrations could play a critical role in maintaining a balanced inflammatory response in early pregnancy to prevent fetal immune rejection and promote normal placental development through the regulation of the secretion of critical cytokines and angiogenic growth factors by human decidual cells. Our data suggest that there is an ontogenically determined regulatory 'switch' in T3 responsiveness between the first and second trimesters, and support the notion that the timely and early correction of maternal thyroid dysfunction is critical in influencing pregnancy outcomes. STUDY FUNDING/COMPETING INTEREST(S): This study is funded by Wellbeing of Women (RG/1082/09 to S.Y.C., M.D.K., J.A.F., L.S.L., G.E.L.) and Action Medical Research - Henry Smith Charity (SP4335 to M.D.K., S.Y.C., L.S.L., J.A.F.). The authors have no conflicts of interest to disclose.

Manipulation of PBF/PTTG1IP phosphorylation status; a potential new therapeutic strategy for improving radioiodine uptake in thyroid and other tumors.

CONTEXT: The clinical effectiveness of ablative radioiodine treatment of thyroid tumors is limited by the availability of the sodium iodide symporter (NIS) at the plasma membrane (PM) for uptake of ¹³¹I. A significant proportion of well-differentiated thyroid tumors are unable to concentrate sufficient radioiodine for effective therapy, and in other tumor models such as breast tumors, where radioiodine uptake would be an attractive therapeutic option, uptake is insufficient. OBJECTIVE: Pituitary tumor-transforming gene-binding factor (PBF; PTTG1IP) is overexpressed in multiple cancers and significantly decreases NIS expression at the PM. The goal of this study was to identify a method by which PBF repression of NIS may be overcome in human tumors. RESULTS: Here, we identify PBF as a tyrosine phosphoprotein that specifically binds the proto-oncogene tyrosine protein kinase Src in mass spectrometry, glutathione S-transferase pulldown and coimmunoprecipitation assays. Src induction leads to phosphorylation at PBF residue Y174. Abrogation of this residue results in PM retention and a markedly reduced ability to bind NIS. The Src inhibitor PP1 inhibits PBF phosphorylation in multiple cell lines in vitro, including human primary thyroid cells. Of direct clinical importance to the treatment of thyroid cancer, PP1 stimulates iodide uptake by transfected NIS in TPC1 thyroid carcinoma cells and entirely overcomes PBF repression of iodide uptake in human primary thyroid cells. CONCLUSIONS: We propose that targeting PBF phosphorylation at residue Y174 via tyrosine kinase inhibitors may be a novel therapeutic strategy to enhance the efficacy of ablative radioiodine treatment in thyroid and other endocrine and endocrine-related tumors.

Expression and function of thyroid hormone transporters in the microvillous plasma membrane of human term placental syncytiotrophoblast.

The transplacental passage of thyroid hormones (THs) from mother to fetus in humans has been deduced from observational clinical studies and is important for normal fetoplacental development. To investigate the transporters that regulate TH uptake by syncytiotrophoblast (the primary barrier to maternal-fetal exchange, which lies in direct contact with maternal blood), we isolated the microvillous plasma membrane (MVM) of human term syncytiotrophoblasts. We have demonstrated that MVM vesicles express plasma membrane TH transporter proteins, including system-L (L-type amino acid transporter 1 and CD98), monocarboxylate transporters (MCTs) 8 and 10, organic anion-transporting polypeptides 1A2 and 4A1. We provide the first definitive evidence that the human syncytiotrophoblast MVM is capable of rapid, saturable T(4) and T(3) uptake at similar rates and in a Na(+)-independent manner. These two major forms of THs could not significantly inhibit each others' uptake, suggesting that each is mediated by largely different transporters. No single transporter was noted to play a dominant role in either T(4) or T(3) uptake. Using combinations of transporter inhibitors that had an additive effect on TH uptake, we provide evidence that 67% of saturable T(4) uptake is facilitated by system-L and MCT10 with a minor role played by organic anion-transporting polypeptides, whereas 87% of saturable T(3) uptake is mediated by MCT8 and MCT10. Our data demonstrate that syncytiotrophoblast may control the quantity and forms of THs taken up by the human placenta. Thus, syncytiotrophoblast could be critical in regulating transplacental TH supply from the mother to the fetus.

PTTG-binding factor (PBF) is a novel regulator of the thyroid hormone transporter MCT8.

Within the basolateral membrane of thyroid follicular epithelial cells, two transporter proteins are central to thyroid hormone (TH) biosynthesis and secretion. The sodium iodide symporter (NIS) delivers iodide from the bloodstream into the thyroid, and after TH biosynthesis, monocarboxylate transporter 8 (MCT8) mediates TH secretion from the thyroid gland. Pituitary tumor-transforming gene-binding factor (PBF; PTTG1IP) is a protooncogene that is up-regulated in thyroid cancer and that binds NIS and modulates its subcellular localization and function. We now show that PBF binds MCT8 in vitro, eliciting a marked shift in MCT8 subcellular localization and resulting in a significant reduction in the amount of MCT8 at the plasma membrane as determined by cell surface biotinylation assays. Colocalization and interaction between PBF and Mct8 was also observed in vivo in a mouse model of thyroid-specific PBF overexpression driven by a bovine thyroglobulin (Tg) promoter (PBF-Tg). Thyroidal Mct8 mRNA and protein expression levels were similar to wild-type mice. Critically, however, PBF-Tg mice demonstrated significantly enhanced thyroidal TH accumulation and reduced TH secretion upon TSH stimulation. Importantly, Mct8-knockout mice share this phenotype. These data show that PBF binds and alters the subcellular localization of MCT8 in vitro, with PBF overexpression leading to an accumulation of TH within the thyroid in vivo. Overall, these studies identify PBF as the first protein to interact with the critical TH transporter MCT8 and modulate its function in vivo. Furthermore, alongside NIS repression, PBF may thus represent a new regulator of TH biosynthesis and secretion.

The expression of thyroid hormone transporters in the human fetal cerebral cortex during early development and in N-Tera-2 neurodifferentiation.

Associations of neurological impairment with mutations in the thyroid hormone (TH) transporter, MCT8, and with maternal hypothyroxinaemia, suggest that THs are crucial for human fetal brain development. It has been postulated that TH transporters regulate the cellular supply of THs within the fetal brain during development. This study describes the expression of TH transporters in the human fetal cerebral cortex (7­20 weeks gestation) and during retinoic acid induced neurodifferentiation of the human N-Tera-2 (NT2) cell line, in triiodothyronine (T3) replete and T3-depleted media. Compared with adult cortex, mRNAs encoding OATP1A2, OATP1C1, OATP3A1 variant 2, OATP4A1, LAT2 and CD98 were reduced in fetal cortex at different gestational ages, whilst mRNAs encoding MCT8, MCT10, OATP3A1 variant 1 and LAT1 were similar. From the early first trimester, immunohistochemistry localised MCT8 and MCT10 to the microvasculature and to undifferentiated CNS cells. With neurodifferentiation, NT2 cells demonstrated declining T3 uptake, accompanied by reduced expressions of MCT8, LAT1, CD98 and OATP4A1. T3 depletion significantly reduced MCT10 and LAT2 mRNA expression at specific time points during neurodifferentiation but there were no effects upon T3 uptake, neurodifferentiation marker expression or neurite lengths and branching. MCT8 repression also did not affect NT2 neurodifferentiation. In conclusion, many TH transporters are expressed in the human fetal cerebral cortex from the first trimester, which could regulate cellular TH supply during early development. However, human NT2 neurodifferentiation is not dependent upon T3 or MCT8 and there were no compensatory changes to promote T3 uptake in a T3-depleted environment.

Differential triiodothyronine responsiveness and transport by human cytotrophoblasts from normal and growth-restricted pregnancies.

CONTEXT: Abnormal placentation in human pregnancy is associated with intrauterine fetal growth restriction (IUGR). Our group has previously reported the association between severe IUGR, lower fetal circulating concentrations of thyroid hormones (THs), and altered expression of TH receptors and TH transporters within human placental villi. We postulate that altered TH bioavailability to trophoblasts may contribute to the pathogenesis of IUGR. DESIGN AND OBJECTIVE: Cytotrophoblasts were isolated from normal and IUGR human placentae to compare their responsiveness to T(3) and their capability for T(3) transport. RESULTS: Compared with normal cytotrophoblasts, the viability of IUGR cytotrophoblasts (assessed by methyltetrazoleum assay) was significantly reduced (P < 0.001), whereas apoptosis (assessed using caspase 3/7 activity and M30 immunoreactivity) was significantly increased after T(3) treatment for 48 h (P < 0.001 and P < 0.01, respectively). The secretion of human chorionic gonadotropin was significantly increased by IUGR cytotrophoblasts compared with normal cytotrophoblasts (P < 0.001), independently of T(3) treatment. Net transport of [(125)I]T(3) was 20% higher by IUGR cytotrophoblasts compared with normal cytotrophoblasts (P < 0.001), and this was accompanied by a 2-fold increase in the protein expression of the TH transporter, monocarboxylate transporter 8, as assessed by Western immunoblotting (P < 0.01). CONCLUSIONS: IUGR cytotrophoblasts demonstrate altered responsiveness to T(3) with significant effects on cell survival and apoptosis compared with normal cytotrophoblasts. Increased monocarboxylate transporter 8 expression and intracellular T(3) accumulation may contribute to the altered T(3) responsiveness of IUGR cytotrophoblasts.

Older subjects with hyperthyroidism present with a paucity of symptoms and signs: a large cross-sectional study.

CONTEXT: The absence of classical symptoms and signs of hyperthyroidism often results in delayed diagnosis and treatment. OBJECTIVES: The objective of the study was to determine the prevalence of symptoms and signs of hyperthyroidism according to patients' age and gender as well as severity and type of hyperthyroidism. DESIGN, PARTICIPANTS AND SETTING: This was a cross-sectional study of 3049 consecutive patients with hyperthyroidism presenting to a single secondary/tertiary care clinic. MAIN OUTCOME MEASURES: Calculation of adjusted odds ratios for presence/absence of symptoms/signs of hyperthyroidism simultaneously analyzing the influence of patients' age/gender, disease etiology/severity, symptom duration, and smoking. RESULTS: The majority of patients older than 61 yr had two or more symptoms. The lowest proportion of subjects reporting five or more symptoms was found in those older than 61 yr. Increasing age was associated with reduced adjusted odds ratio for the presence of most classical symptoms except for weight loss and shortness of breath, independent of disease severity. Those with more severe hyperthyroidism and smokers had increased odds ratios for most symptoms. Older age, higher serum free T(4) concentrations at diagnosis, male gender, and toxic nodular hyperthyroidism were independently associated with risk of atrial fibrillation. Signs of ophthalmopathy were associated with increasing age, smoking, longer symptom duration, and female gender. CONCLUSIONS: Classical symptoms and signs of hyperthyroidism are significantly less prevalent in older patients and more prevalent in smokers and subjects with higher free T(4) concentrations. We propose a lower threshold for performing thyroid function tests in patients older than 60 yr, especially in those presenting with atrial fibrillation, weight loss, or shortness of breath.

Expression of thyroid hormone transporters in the human placenta and changes associated with intrauterine growth restriction.

Thyroid hormones (TH) are important for the development of the human fetus and placenta from very early gestation. The transplacental passage of TH from mother to fetus and the supply of TH into trophoblasts require the expression of placental TH plasma membrane transporters. We describe the ontogeny of the TH transporters MCT8, MCT10, LAT1, LAT2, OATP1A2 and OATP4A1 in a large series (n = 110) of normal human placentae across gestation and describe their expression changes with intrauterine fetal growth restriction (IUGR n = 22). Quantitative RT-PCR revealed that all the mRNAs encoding TH transporters are expressed in human placenta from 6 weeks gestation and throughout pregnancy. MCT8, MCT10, OATP1A2 and LAT1 mRNA expression increased with gestation. OATP4A1 and CD98 (LATs obligatory associated protein) mRNA expression reached a nadir in mid-gestation before increasing towards term. LAT2 mRNA expression did not alter throughout gestation. Immunohistochemistry localised MCT10 and OATP1A2 to villous cytotrophoblasts and syncytiotrophoblasts, and extravillous trophoblasts while OATP4A1 was preferentially expressed in the villous syncytiotrophoblasts. Whilst MCT8 protein expression was increased, MCT10 mRNA expression was decreased in placentae from IUGR pregnancies delivered in the early 3rd trimester compared to age matched appropriately grown for gestational age controls. No significant change was found in the mRNA expression of the other transporters with IUGR. In conclusion, several TH transporters are present in the human placenta from early 1st trimester with varying patterns of expression throughout gestation. Their coordinated effects may regulate both transplacental TH passage and TH supply to trophoblasts, which are critical for the normal development of the fetus and placenta. Increased MCT8 and decreased MCT10 expression within placentae of pregnancies complicated by IUGR may contribute to aberrant development of the fetoplacental unit.

Monocarboxylate transporter 8 in neuronal cell growth.

Thyroid hormones are essential for the normal growth and development of the fetus, and even small alterations in maternal thyroid hormone status during early pregnancy may be associated with neurodevelopmental abnormalities in childhood. Mutations in the novel and specific thyroid hormone transporter monocarboxylate transporter 8 (MCT8) have been associated with severe neurodevelopmental impairment. However, the mechanism by which MCT8 influences neural development remains poorly defined. We have therefore investigated the effect of wild-type (WT) MCT8, and the previously reported L471P mutant, on the growth and function of human neuronal precursor NT2 cells as well as MCT8-null JEG-3 cells. HA-tagged WT MCT8 correctly localized to the plasma membrane in NT2 cells and increased T(3) uptake in both cell types. In contrast, L471P MCT8 was largely retained in the endoplasmic reticulum and displayed no T(3) transport activity. Transient overexpression of WT and mutant MCT8 proteins failed to induce endoplasmic reticular stress or apoptosis. However, MCT8 overexpression significantly repressed cell proliferation in each cell type in both the presence and absence of the active thyroid hormone T(3) and in a dose-dependent manner. In contrast, L471P MCT8 showed no such influence. Finally, small interfering RNA depletion of endogenous MCT8 resulted in increased cell survival and decreased T(3) uptake. Given that T(3) stimulated proliferation in embryonic neuronal NT2 cells, whereas MCT8 repressed cell growth, these data suggest an entirely novel role for MCT8 in addition to T(3) transport, mediated through the modulation of cell proliferation in the developing brain.

Prediction of cure and risk of hypothyroidism in patients receiving 131I for hyperthyroidism.

CONTEXT: There is little consensus regarding the most appropriate dose of radioiodine ((131)I) to be administered to patients with hyperthyroidism. OBJECTIVE: To compare the efficacy of fixed dose regimens of (131)I in curing hyperthyroidism and to define simple clinical and biochemical factors that predict outcome in individual patients. DESIGN: Consecutive series of hyperthyroid subjects treated with (131)I. SETTING: Single Secondary/Tertiary Care Hospital Clinic. PARTICIPANTS: A total of 1278 patients (1013 females and 262 males, mean age 49.7 years) presenting with hyperthyroidism between 1984 and 2006. INTERVENTION: Treatment with (131)I using a fixed dose regimen. MAIN OUTCOME MEASURES: Probability of cure and risk of development of hypothyroidism following a single dose of (131)I. RESULTS: Patients given a single dose of (131)I of 600 MBq (n = 485) had a higher cure rate (84.1%) compared with those receiving either 370 MBq (74.9%, P < 0.001) or those given 185 Bq (63%, P < 0.001). An increased incidence of hypothyroidism by 1 year was evident with higher doses (600 MBq: 60.4%; 370 MBq: 49.2%, P = 0.001; 185 Bq: 38.1%, P < 0.001). Binary logistic regression analysis identified a 600 Bq dose of (131)I [adjusted odds ratio, AOR 3.33 (2.28-4.85), P < 0.001], female gender [AOR 1.75 (1.23-2.47), P = 0.002], lower presenting serum free T4 concentration [AOR 1.01 (1.01-1.02), P < 0.001] and absence of a palpable goitre [AOR 3.33 (2.00-5.56), P < 0.001] to be independent predictors of cure. Similarly, a 600 MBq dose [AOR 3.79 (2.66-5.38), P < 0.001], female gender [AOR 1.46 (1.05-2.02), P = 0.02], younger age [AOR 1.03 (1.02-1.04), P < 0.001], absence of a palpable goitre [AOR 3.85 (2.38-5.88), P < 0.001] and presence of ophthalmopathy [AOR 1.57 (1.06-2.31), P = 0.02] were identified as independent factors predicting the probability of development of hypothyroidism at one year. Based on these findings, formulae to indicate probability of cure and risk of hypothyroidism for application to individual patients were derived. CONCLUSIONS: Simple clinical/biochemical criteria can be used to predict outcome after (131)I treatment. These factors determine that males, those with severe biochemical hyperthyroidism, and those with a palpable goitre require larger doses (600 MBq) in order to achieve cure.

Analysis of HLA class II genes in Hashimoto's thyroiditis reveals differences compared to Graves' disease.

Graves' disease (GD) and Hashimoto's thyroiditis (HT) represent the commonest forms of autoimmune thyroid disease (AITD) each presenting with distinct clinical features. Progress has been made in determining association of HLA class II DRB1, DQB1 and DQA1 loci with GD demonstrating a predisposing effect for DR3 (DRB1(*)03-DQB1(*)02-DQA1(*)05) and a protective effect for DR7 (DRB1(*)07-DQB1(*)02-DQA1(*)02). Small data sets have hindered progress in determining HLA class II associations with HT. The aim of this study was to investigate DRB1-DQB1-DQA1 in the largest UK Caucasian HT case control cohort to date comprising 640 HT patients and 621 controls. A strong association between HT and DR4 (DRB1(*)04-DQB1(*)03-DQA1(*)03) was detected (P=6.79 x 10(-7), OR=1.98 (95% CI=1.51-2.59)); however, only borderline association of DR3 was found (P=0.050). Protective effects were also detected for DR13 (DRB1(*)13-DQB1(*)06-DQA1(*)01) (P=0.001, OR=0.61 (95% CI=0.45-0.83)) and DR7 (P=0.013, OR=0.70 (95% CI=0.53-0.93)). Analysis of our unique cohort of subjects with well characterized AITD has demonstrated clear differences in association within the HLA class II region between HT and GD. Although HT and GD share a number of common genetic markers this study supports the suggestion that differences in HLA class II genotype may, in part, contribute to the different immunopathological processes and clinical presentation of these related diseases.

Subclinical thyroid disorders--consequences and implications for treatment.

Subclinical hyperthyroidism is characterized by low circulating TSH concentrations, together with normal concentrations of free T4 and free T3. The clinical significance of low but detectable TSH values is likely to be different from undetectable TSH values. The question remains as to whether endogenous and exogenous (iatrogenic) causes of low serum TSH have the same clinical significance. Subclinical hypothyroidism is characterized by high serum TSH concentrations, together with normal free T4 values.

Preliminary evidence for interaction of PTPN12 polymorphism with TSHR genotype and association with Graves' ophthalmopathy.

OBJECTIVE: Protein tyrosine phosphatases (PTPs), such as PTPN22, are important regulators of signal transduction from the T cell receptor and have been associated with autoimmunity. PTPN12 interacts with the same T cell activation accessory molecules, Grb2 and Csk kinase, as the Graves' disease (GD) associated PTPN22 encoded lymphoid tyrosine phosphatase (LYP) molecule and also plays a key role in T cell receptor signalling, leading to the hypothesis that it too may be involved in GD susceptibility. DESIGN: PTPN12 was tested for association in a large well-characterized UK Caucasian case control cohort using seven tagging single nucleotide polymorphisms (SNPs). Patients A total of 1058 GD patients and 864 controls. Measurements Tests for association with the disease. RESULTS: Despite adequate statistical power to detect an effect if present, none of the seven tag SNPs were associated with GD (P = 0.925-0.089). Three SNPs (rs1468682, rs4729535 and rs17467232), however, demonstrated association with the presence of ophthalmopathy NOSPECS classes 2-4 (P = 0.039-0.004). Four SNPs (rs1468682, rs4729535, rs17155601 and rs17467232) revealed evidence of interaction with the previously associated thyrotropin hormone receptor (TSHR) rs2268458 SNP (P = 0.035-0.002). CONCLUSIONS: No association was detected between individual PTPN12 tag SNPs and GD but preliminary evidence suggests PTPN12 confers an increased risk of mild/moderate ophthalmopathy (NOSPECS classes 2-4) and that PTPN12 interacts with the TSHR. Replication of these preliminary results is now required in larger independent datasets to validate these findings.

Tag SNP screening of the PDCD1 gene for association with Graves' disease.

OBJECTIVE: The Programmed Cell Death 1 gene (PDCD1) on chromosome 2q37.3 encodes PD-1 which is involved in providing a negative signal to activated T cells. Large case-control studies have shown association of PDCD1 with several autoimmune diseases although, to date, no such studies have been performed for Graves' disease (GD). The objective of our study was to investigate eight tag SNPs representing the majority of common variation in PDCD1 within a well-characterized large UK Caucasian GD dataset. DESIGN: A case control association study of eight polymorphisms. PATIENTS: 2671 Graves' disease patients and 864 controls. MEASUREMENTS: Tests for association with disease. RESULTS: No association with disease was seen for any of the +4163, +5049, +5318, +5640, +5678 and +7078 SNPs genotyped in this study. Association was detected between the +2375 SNP (P = 0.021, OR = 1.14 [95% CI = 1.01-1.29]) and GD and a small protective effect was seen with the +6799 SNP genotypes (P = 0.028, OR = 0.77 [95% CI = 0.58-1.03]). CONCLUSIONS: This study has, for the first time, shown that small effects within PDCD1 may contribute towards the development of GD, supporting the hypothesis that much of the currently unknown genetic contribution to GD could be due to several small genetic effects with ORs 1.2. Replication of this result is now needed to confirm our findings and justify more detailed fine mapping of a primary aetiological variant in this gene region.

Association between serum free thyroxine concentration and atrial fibrillation.

BACKGROUND: Previous studies have suggested that minor changes in thyroid function are associated with risk of atrial fibrillation (AF). Our objective was to determine the relationship between thyroid function and presence of atrial fibrillation (AF) in older subjects. METHODS: A population-based study of 5860 subjects 65 years and older, which excluded those being treated for thyroid dysfunction and those with previous hyperthyroidism. Main outcome measures included tests of thyroid function (serum free thyroxine [T(4)] and thyrotropin [TSH]) and the presence of AF on resting electrocardiogram. RESULTS: Fourteen subjects (0.2%) had previously undiagnosed overt hyperthyroidism and 126 (2.2%), subclinical hyperthyroidism; 5519 (94.4%) were euthyroid; and 167 (2.9%) had subclinical hypothyroidism and 23 (0.4%), overt hypothyroidism. The prevalence of AF in the whole cohort was 6.6% in men and 3.1% in women (odds ratio, 2.23; P<.001). After adjusting for sex, logistic regression showed a higher prevalence of AF in those with subclinical hyperthyroidism compared with euthyroid subjects (9.5% vs 4.7%; adjusted odds ratio, 2.27; P=.01). Median serum free T(4) concentration was higher in those with AF than in those without (1.14 ng/dL; interquartile range [IQR], 1.05-1.27 ng/dL [14.7 pmol/L; IQR, 13.5-16.4 pmol/L] vs 1.10 ng/dL; IQR, 1.00-1.22 ng/dL [14.2 pmol/L; IQR, 12.9-15.7 pmol/L]; P<.001), and higher in those with AF when analysis was limited to euthyroid subjects (1.13 ng/dL; IQR, 1.05-1.26 ng/dL [14.6 pmol/L; IQR, 13.5-16.2 pmol/L] vs 1.10 ng/dL; IQR, 1.01-1.21 ng/dL [14.2 pmol/L; IQR, 13.0-15.6 pmol/L]; P=.001). Logistic regression showed serum free T(4) concentration, increasing category of age, and male sex all to be independently associated with AF. Similar independent associations were observed when analysis was confined to euthyroid subjects with normal TSH values. CONCLUSIONS: The biochemical finding of subclinical hyperthyroidism is associated with AF on resting electrocardiogram. Even in euthyroid subjects with normal serum TSH levels, serum free T(4) concentration is independently associated with AF.

Separase, securin and Rad21 in neural cell growth.

The key mitotic regulator securin is expressed at low levels in fetal brain compared with adult, and modulates the proliferation of human embryonic neuronal N-Tera2 (NT2) cells. We now examine the function and expression of securin's interacting partner separase, along with Rad21, the functional component of cohesin, which is cleaved by separase following interaction with securin. In contrast to securin, the cleaved forms of separase and Rad21 were highly expressed in human fetal cerebral cortex compared with adult. In a murine model of absent securin expression - the PTTG knock-out mouse - separase and Rad21 were over-expressed in multiple brain regions. In addition, cDNA array analysis of other key mitotic regulators additionally identified cyclin C and sestrin 2 to be induced in the brains of securin-null mice compared with wild type. Further, Rad21 mRNA expression was highly correlated with that of securin, separase, cyclin C and sestrin 2 in fetal brains. In embryonic neuronal NT2 cells, siRNA repression of separase failed to significantly alter cell turnover, whereas repression of securin expression resulted in increased levels of the activated forms of Rad21 and separase, and promoted cell proliferation. Our data suggest that the co-ordinated expression of separase, securin and Rad21 is fundamental for the developing brain.

PTTG and PBF repress the human sodium iodide symporter.

The ability of the thyroid to accumulate iodide provides the basis for radioiodine ablation of differentiated thyroid cancers and their metastases. Most thyroid tumours exhibit reduced iodide uptake, although the mechanisms accounting for this remain poorly understood. Pituitary tumour transforming gene (PTTG) is a proto-oncogene implicated in the pathogenesis of thyroid tumours. We now show that PTTG and its binding factor PBF repress expression of sodium iodide symporter (NIS) messenger RNA (mRNA), and inhibit iodide uptake. This process is mediated at least in part through fibroblast growth factor-2. In detailed studies of the NIS promoter in rat FRTL-5 cells, PTTG and PBF demonstrated specific inhibition of promoter activity via the human upstream enhancer element (hNUE). Within this approximately 1 kb element, a complex PAX8-upstream stimulating factor 1 (USF1) response element proved critical both to basal promoter activity and to PTTG and PBF repression of NIS. In particular, repression by PTTG was contingent upon the USF1, but not the PAX8, site. Finally, in human primary thyroid cells, PTTG and PBF similarly repressed the NIS promoter via hNUE. Taken together, our data suggest that the reported overexpression of PTTG and PBF in differentiated thyroid cancer has profound implications for activity of the NIS gene, and hence significantly impacts upon the efficacy of radioiodine treatment.

Securin induces genetic instability in colorectal cancer by inhibiting double-stranded DNA repair activity.

Genetic instability (GI) is a hallmark feature of tumor development. Securin, also known as pituitary tumor transforming gene (PTTG), is a mitotic checkpoint protein which is highly expressed in numerous cancers, is associated with tumor invasiveness, and induces GI in thyroid cells. We used fluorescence inter-simple sequence repeat PCR to assess GI caused primarily by DNA breakage events in 19 colorectal tumors. GI values ranged significantly, with Dukes' stage C&D colorectal tumors exhibiting greater GI and higher securin expression than Dukes' stage A&B tumors. Consistent with these findings, we observed a dose-dependent increase in GI in HCT116 cells in response to securin overexpression, as well as in non-transformed human fibroblasts. As securin has been implicated in a novel DNA repair pathway in fission yeast, we investigated its potential role in chemotoxic DNA damage response pathways in mammalian cells, using host cell reactivation assays. Securin overexpression in HCT116 cells inhibited etoposide-induced double-stranded DNA damage repair activity, and repressed Ku heterodimer function. Additionally, we observed that securin and Ku70 showed a reciprocal cytosol-nuclear translocation in response to etoposide-induced dsDNA damage. Our data suggest that, by repressing Ku70 activity and inhibiting the non-homologous end-joining dsDNA repair pathway, securin may be a critical gene in the development of GI in colorectal cancer.

Association of the BTNL2 rs2076530 single nucleotide polymorphism with Graves' disease appears to be secondary to DRB1 exon 2 position beta74.

OBJECTIVE: The HLA region encodes numerous immune response genes, with the DR/DQ molecules consistently associated with autoimmune disease (AID). Recent studies in sarcoidosis have identified association of a single nucleotide polymorphism (SNP) rs2076530 within BTNL2, a potential T-cell inhibitor, independent of the known DRB1 association. The aim of this study was to investigate the association rs2076530 with disease in a large UK Caucasian Graves' disease (GD) dataset. DESIGN: A case control association study of the rs2076530 polymorphism. PATIENTS: Eight hundred sixty-four Graves' disease patients and 864 controls. MEASUREMENTS: Tests for association with disease. RESULTS: We detected association of rs2076530 within a large GD dataset [OR = 1.32 (95% CI = 1.14-1.52)], however, linkage disequilibrium (LD) analysis revealed association of rs2076530 to be secondary to the previously established DRB1 exon 2 encoded position beta74 effect although a rare haplotype effect, including both loci, cannot be excluded. CONCLUSIONS: BTNL2 may be a sarcoidosis-specific susceptibility loci, although only extensive examination of the whole HLA region in different inflammatory/AIDs will enable DR/DQ independent HLA effects to be determined.

Influences of age, gender, smoking, and family history on autoimmune thyroid disease phenotype.

CONTEXT: Both genetic and environmental factors contribute to susceptibility to Graves' disease (GD) and Hashimoto's thyroiditis (HT), as well as disease manifestations. OBJECTIVE: The objective of the study was to define how endogenous/environmental factors contribute to variation in phenotype. DESIGN/SETTING: This was a multicenter cohort study. PATIENTS/OUTCOME MEASURES: We prospectively collected clinical/biochemical data as part of the protocol for a United Kingdom DNA collection for GD and HT. We investigated, in 2805 Caucasian subjects, whether age at diagnosis, gender, family history (FH), smoking history, and presence of goiter influenced disease manifestations. RESULTS: For 2405 subjects with GD, the presence of goiter was independently associated with disease severity (serum free T4 at diagnosis) (P < 0.001). Free T4 (P < 0.05) and current smoking (P < 0.001) were both independent predictors of the presence of ophthalmopathy. Approximately half of those with GD (47.4% of females, 40.0% of males) and HT (n = 400) (56.4% of females, 51.7% of males) reported a FH of thyroid dysfunction. In GD, a FH of hyperthyroidism in any relative was more frequent than hypothyroidism (30.1 vs. 24.4% in affected females, P < 0.001). In HT, a FH of hypothyroidism was more common than hyperthyroidism (42.1 vs. 22.8% in affected females, P < 0.001). For GD (P < 0.001) and HT (P < 0.05), a FH was more common in maternal than paternal relatives. The reporting of a parent with thyroid dysfunction (hyper or hypo) was associated with lower median age at diagnosis of both GD (mother with hyperthyroidism, P < 0.001) and HT (father with hypothyroidism, P < 0.05). In GD and HT, there was an inverse relationship between the number of relatives with thyroid dysfunction and age at diagnosis (P < 0.01). CONCLUSIONS: Marked associations among age at diagnosis, disease severity, goiter, ophthalmopathy, smoking, and FH provide evidence for interactions between genetic and environmental/endogenous factors; understanding these may allow preventive measures or better tailoring of therapies.