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Bezafibrate (BZ) regulates mitochondrial biogenesis by activation of PPAR's receptors and enhancing the level of PGC-1α coactivator. In this report, we investigated the effect of BZ on the expression of genes (1) that are linked to different pathways involved in mitochondrial biogenesis, e.g., regulated by PPAR's receptors or PGC-1α coactivator, and (2) involved in neuronal or astroglial fate, during neural differentiation of hiPSC. The tested cell populations included hiPSC-derived neural stem cells (NSC), early neural progenitors (eNP), and neural progenitors (NP). RNA-seq analysis showed the expression of PPARA, PPARD receptors and excluded PPARG in all tested populations. The expression of PPARGC1A encoding PGC-1α was dependent on the stage of differentiation: NSC, eNP, and NP differed significantly as compared to hiPSC. In addition, BZ-evoked upregulation of PPARGC1A, GFAP, S100B, and DCX genes coexist with downregulation of MAP2 gene only at the eNP stage of differentiation. In the second task, we investigated the cell sensitivity and mitochondrial biogenesis upon BZ treatment. BZ influenced the cell viability, ROS level, mitochondrial membrane potential, and total cell number in concentration- and stage of differentiation-dependent manner. Induction of mitochondrial biogenesis evoked by BZ determined by the changes in the level of SDHA and COX-1 protein, and mtDNA copy number, as well as the expression of NRF1, PPARGC1A, and TFAM genes, was detected only at NP stage for all tested markers. Thus, developmental stage-specific sensitivity to BZ of neurally differentiating hiPSC can be linked to mitochondrial biogenesis, while fate commitment decisions to PGC-1α (encoded by PPARGC1A) pathway.

Amphipod crustaceans were collected at all 55 stations sampled with an epibenthic sledge during two IceAGE expeditions (Icelandic marine Animals: Genetics and Ecology) in 2011 and 2013. In total, 34 amphipod families and three superfamilies were recorded in the samples. Distribution maps are presented for each taxon along with a summary of the regional taxonomy for the group. Statistical analyses based on presence/absence data revealed a pattern of family distributions that correlated with sampling depth. Clustering according to the geographic location of the stations (northernmost North Atlantic Sea and Arctic Ocean) can also be observed. IceAGE data for the Amphilochidae and Oedicerotidae were analysed on species level; in case of the Amphilochidae they were compared to the findings from a previous Icelandic benthic survey, BIOICE (Benthic Invertebrates of Icelandic waters), which also identified a high abundance of amphipod fauna.

Multiple factors underlie the pathophysiology of hypertension, involving endothelial dysregulation, vascular smooth muscle dysfunction, increased oxidative stress, sympathetic nervous system activation and altered renin -angiotensin -aldosterone regulatory activity. A class of non-coding RNA called microRNA, consisting of 17-25 nucleotides, exert regulatory function over these processes. This paper summarizes the currently available data from preclinical and clinical studies on miRNA in the development of hypertension as well as the impact of anti-hypertensive treatment on their plasma expression. We present microRNAs' characteristics, their biogenesis and role in the regulation of blood pressure together with their potential diagnostic and therapeutic application in clinical practice.

Papillary Thyroid Carcinoma (PTC) displays one of the highest familiality scores of all cancers as measured by case-control studies, yet only a handful of genes have been implicated until now. Variants in microRNAs have been associated with the risk of several cancers including PTC but the magnitude of this involvement is unclear. This study was designed to test to what extent genomic variants in microRNAs contribute to PTC risk. We used SOLiD technology to sequence 321 genomic regions encoding 427 miRNAs in one affected individual from each of 80 PTC families. After excluding variants with frequency ≥ 1% in 1000 Genomes Phase 1 (n = 1092) we detected 1978 variants. After further functional filtering steps 25 variants in pre-miRs remained. Co-segregation was observed for six out of 16 tested miRNA variants with PTC in the families, namely let-7e, miR-181b, miR-135a, miR-15b, miR-320, and miR-484. Expression of miR-135a and miR-181b was tested in normal thyroid and tumor tissue from patients that carry the variants and a decrease in expression was observed. In vitro assays were applied to measure the effect of the variants on microRNAs' maturation. Four out of six variants were tested. Only the let-7e and miR-181b variants showed an effect on processing leading to lower levels of mature miRNA. These two variants were not detected in 1170 sporadic PTC cases nor in 1404 controls. Taken together, our data show that high penetrance germline sequence variants of miRNAs potentially predispose to a fraction of all PTC but are not common.

Na(+)/I(-) symporter (NIS)-mediated radioiodide uptake (RAIU) serves as the basis for targeted ablation of thyroid cancer remnants. However, many patients with thyroid cancer have reduced NIS expression/function and hence do not benefit from radioiodine therapy. microRNA (miR) has emerged as a promising therapeutic target in many diseases; yet, the role of miRs in NIS-mediated RAIU has not been investigated. In silico analysis was used to identify miRs that may bind to the 3'UTR of human NIS (hNIS). The top candidate miR-339-5p directly bound to the 3'UTR of hNIS. miR-339-5p overexpression decreased NIS-mediated RAIU in HEK293 cells expressing exogenous hNIS, decreased the levels of NIS mRNA, and RAIU in transretinoic acid/hydrocortisone (tRA/H)-treated MCF-7 human breast cancer cells as well as thyrotropin-stimulated PCCl3 rat thyroid cells. Nanostring nCounter rat miR expression assay was conducted to identify miRs deregulated by TGFβ, Akti-1/2, or 17-AAG known to modulate RAIU in PCCl3 cells. Among 38 miRs identified, 18 were conserved in humans. One of the 18 miRs, miR-195, was predicted to bind to the 3'UTR of hNIS and its overexpression decreased RAIU in tRA/H-treated MCF-7 cells. miR-339-5p was modestly increased in most papillary thyroid carcinomas (PTCs), yet miR-195 was significantly decreased in PTCs. Interestingly, the expression profiles of 18 miRs could be used to distinguish most PTCs from nonmalignant thyroid tissues. This is the first report, to our knowledge, demonstrating that hNIS-mediated RAIU can be modulated by miRs, and that the same miRs may also play roles in the development or maintenance of thyroid malignancy. Accordingly, miRs may serve as emerging targets to halt the progression of thyroid cancer and to enhance the efficacy of radioiodine therapy., (© 2015 Society for Endocrinology.)

Hepatocellular carcinoma (HCC) represents the major histological subtype of liver cancer. Tumorigenic changes in hepatic cells potentially result from aberrant expression of microRNAs (miRNAs). Individual microRNA gene may give rise to miRNAs of different length, named isomiRNAs that proved to be functionally relevant. Since microRNA length heterogeneity in hepatic tissue has not been described before, we employed next-generation sequencing to comprehensively analyze microRNA transcriptome in HCC tumors (n=24) and unaffected tissue adjacent to tumors (n=24), including samples with (n=15) and without cirrhosis (n=9). We detected 374 microRNAs expressed in liver, including miR-122-5p that constituted over 39% of the hepatic miRnome. Among the liver expressed miRs, the levels of 64 significantly differed between tumor and control samples (FDR<0.05, fold change>2). Top deregulated miRNAs included miR-1269a (T/N=22.95), miR-3144-3p (T/N=5.24), miR-183-5p (T/N=4.63), miR-10b-5p (T/N=3.87), miR-490-3p (T/N=0.13), miR-199a-5p (T/N=0.17), miR-199a-3p/miR-199b-3p (T/N=0.19), miR-214-5p (T/N=0.20) and miR-214-3p (T/N=0.21). Almost all miRNA genes produced several mature molecules differing in length (isomiRNAs). The reference sequence was not the most prevalent in 38.6% and completely absent in 10.5% of isomiRNAs. Over 26.1% of miRNAs produced isoforms carrying≥2 alternative seed regions, of which 35.5% constituted novel, previously unknown seeds. This fact sheds new light on the percentage of the human genome regulated by microRNAs and their variants. Among the most deregulated miRNAs, miR-199a-3p/miR-199b-3p (T/N fold change=0.18, FDR=0.005) was expressed in 9 isoforms with 3 different seeds, concertedly leading to upregulation of TGF-beta signaling pathway (OR=1.99; p=0.004). In conclusion, the study reveals the comprehensive miRNome of hepatic tissue and provides new tools for investigation of microRNA-dependent pathways in cirrhotic liver and hepatocellular carcinoma. This article is part of a Directed Issue entitled: Rare Cancers., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Two recent genome-wide association studies (GWASs) identified five single nucleotide polymorphisms (SNPs; rs965513, rs944289, rs966423, rs2439302, and rs116909374) associated with papillary thyroid carcinoma (PTC). Each variant showed highly significant but moderate to low disease risk. Here we assessed the cumulative risk and predictive value of the five SNPs., Methods: We genotyped two cohorts of individuals, 747 PTC cases and 1047 controls from Ohio and 1795 PTC cases and 2090 controls from Poland. Cumulative genetic risk scores were calculated using unweighted and weighted approaches., Results: All five SNPs showed significant association with PTC. The average cumulative risk score in cases was significantly higher than in controls (p<2.2×10(-16)). Each additional risk allele increased the risk of having PTC by 1.51 [95% confidence interval (CI) 1.4, 1.64] in Ohio and by 1.35 [95% CI 1.27, 1.44] in Poland. An analysis was performed weighing risk alleles by effect size and assigning individuals to three weighted risk score groups, low (≤2), medium (2-5), and high (>5). Individuals in the high group were significantly more susceptible to PTC compared with individuals in the low group with an odds ratio of 8.7 [95% CI 5.8, 13.3] in Ohio and 4.24 [95% CI 3.10, 5.84] in Poland. Almost identical results were obtained when follicular variant PTCs and microPTCs were omitted. These five SNPs explained 11% of the familial risk of thyroid cancer in the Ohio cohort and 6% in the Polish cohort., Conclusion: As the genetic risk score increases, the risk of having PTC increases. However, the predictive power of the cumulative effect of these five variants is only moderately high and clinical use may not be feasible until more variants are detected.

Context: A single microRNA gene may give rise to several mature products that differ in length, called isomiRs. IsomiRs are known to be tissue specific and functionally relevant. The microRNA sequence heterogeneity of the thyroid gland has yet to be determined., Objective: The objective of the study was to provide a comprehensive view of the microRNA transcriptome in normal thyroid and papillary thyroid carcinoma (PTC)., Design: We used next-generation deep sequencing to analyze microRNA length heterogeneity and expression profiles of PTC tumors (n = 14), unaffected tissue adjacent to tumors (n = 14), and control, noncancerous thyroid tissue (n = 14). The results were validated with a microarray on an additional set of 9 PTC tumor/normal tissue pairs., Results: Eighty-nine microRNAs were significantly deregulated in PTC compared with normal thyroid tissue (false discovery rate < 0.05, fold change 0.13-20.7). Top deregulated miRNAs included miR-146b-5p, miR-221-3p, miR-7-3p, miR-551b-3p, miR-486-3p, and miR-144-3p, confirming previous microarray profiling. The expression of miRNAs did not depend on the BRAF mutation status. Interestingly, 85% of the most abundant microRNAs consisted of isoforms that differed from the standard reference sequence deposited in miRBase. Moreover, the reference microRNAs were completely absent in 42.4% and 35.9% of the microRNAs expressed in normal thyroid and PTC tumors, respectively. Numerous isomiRs had altered seed sequences, which led to a different set of target genes. For highly deregulated miR-146b-5p, we detected 6 isoforms (tumor/normal fold change 14.4-28.7, false discovery rate < 0.002) that varied at their 5' ends with a 1-nt difference that created 2 alternative seeds. The target genes for those 2 seeds overlapped in only 13.1% of genes., Conclusions: Almost all microRNAs exhibit isoforms of variable length and potentially distinct function in thyroid tumorigenesis.

Thyroid cancer shows high heritability but causative genes remain largely unknown. According to a common hypothesis the genetic predisposition to thyroid cancer is highly heterogeneous; being in part due to many different rare alleles. Here we used linkage analysis and targeted deep sequencing to detect a novel single-nucleotide mutation in chromosome 4q32 (4q32A>C) in a large pedigree displaying non-medullary thyroid carcinoma (NMTC). This mutation is generally ultra-rare; it was not found in 38 NMTC families, in 2676 sporadic NMTC cases or 2470 controls. The mutation is located in a long-range enhancer element whose ability to bind the transcription factors POU2F and YY1 is significantly impaired, with decreased activity in the presence of the C- allele compared with the wild type A-allele. An enhancer RNA (eRNA) is transcribed in thyroid tissue from this region and is greatly downregulated in NMTC tumors. We suggest that this is an example of an ultra-rare mutation predisposing to thyroid cancer with high penetrance.

Background: Papillary thyroid carcinoma (PTC) shows high heritability, yet efforts to find predisposing genes have been largely negative., Objectives: The objective of this study was to identify susceptibility genes for PTC., Methods: A genome-wide linkage analysis was performed in 38 families. Targeted association study and screening were performed in 2 large cohorts of PTC patients and controls. Candidate DNA variants were tested in functional studies., Results: Linkage analysis and association studies identified the Slit-Robo Rho GTPase activating protein 1 gene (SRGAP1) in the linkage peak as a candidate gene. Two missense variants, Q149H and A275T, localized in the Fes/CIP4 homology domain segregated with the disease in 1 family each. One missense variant, R617C, located in the RhoGAP domain occurred in 1 family. Biochemical assays demonstrated that the ability to inactivate CDC42, a key function of SRGAP1, was severely impaired by the Q149H and R617C variants., Conclusions: Our findings suggest that SRGAP1 is a candidate gene in PTC susceptibility. SRGAP1 is likely a low-penetrant gene, possibly of a modifier type.

Context: Traditionally, factors predisposing to diseases are either genetic ("nature") or environmental, also known as lifestyle-related ("nurture"). Papillary thyroid cancer is an example of a disease where the respective roles of these factors are surprisingly unclear., Evidence Acquisition: Original articles and reviews summarizing our current understanding of the role of microRNA in thyroid tumorigenesis are reviewed and evaluated., Conclusion: The genetic predisposition to papillary thyroid cancer appears to consist of a variety of gene mutations that are mostly either of low penetrance and common or of high penetrance but rare. Moreover, they likely interact with each other and with environmental factors. The culpable genes may not be of the traditional, protein-coding type. A limited number of noncoding candidate genes have indeed been described, and we propose here that the failure to find mutations in traditional protein-coding genes is not coincidental. Instead, a more likely hypothesis is that changes in the expression of multiple regulatory RNA genes, e.g. microRNAs, may be a major mechanism. Our review of the literature strongly supports this notion in that a polymorphism in one microRNAs (miR-146a) predisposes to thyroid carcinoma, whereas numerous other microRNAs are involved in signaling (mainly PTEN/PI3K/AKT and T3/THRB) that is central to thyroid carcinogenesis.

Context: Loss of the thyroid hormone receptor is common in tumors. In mouse models, a truncated THRB gene leads to thyroid cancer. Previously, we observed up-regulation of the expression of eight microRNAs (miRs) in papillary thyroid carcinoma (PTC) tumors., Objective: Our objective was to determine whether THRB might be inhibited by miRs up-regulated in PTC., Design: The potential binding of miR to the 3'-untranslated region of THRB was analyzed in silico. Direct inhibition by miRs binding to the cloned 3'-untranslated region of THRB was evaluated using luciferase assays. Inhibition of endogenous THRB and its target genes (DIO1 and APP) was examined in cell lines transfected by pre-miRs. The impact on thyroid hormone response element (TRE) was evaluated in promoter assays. Correlations between the expression of THRB and miRs was evaluated in 13 PTC tumor/normal tissue pairs., Results: THRB contains binding sites for the top seven miRs up-regulated in PTC (P = 0.0000002). Direct interaction with THRB was shown for miR-21 and miR-146a. We observed lower levels of THRB transcripts in cell lines transfected with miR-21, -146a, and -221 (down-regulation of 37-48%; P < 0.0001), but not with miR-181a. THRB protein was suppressed down to 10-28% by each of four miRs. Concomitant expression of DIO1 and APP was affected (down-regulation of 32-66%, P < 0.0034 and up-regulation of 48-57%, P < 0.0002, respectively). All four miRs affected TRE activity in promoter assays. Down-regulation of luciferase occurred after transfection with pTRE-TK-Luc construct and each of four miRs. The analysis of tumor/normal tissue pairs revealed down-regulation of THRB in 11 of 13 pairs (1.3- to 9.1-fold), and up-regulation of miR-21, -146a, -181a, and -221 in almost all pairs., Conclusions: MiRs up-regulated in PTC tumors directly inhibit the expression of THRB, an important tumor suppressor gene.

Prior work has shown that heterozygosity G/C of single nucleotide polymorphism (SNP rs2910164) within the precursor of microRNA-146a predisposes to PTC (odds ratio = 1.62, P = 0.000007) although the mechanism was unclear. Here, we show that GC heterozygotes differ from both GG and CC homozygotes by producing 3 mature microRNAs: 1 from the leading strand (miR-146a), and 2 from the passenger strand (miR-146a*G and miR-146a*C), each with its distinct set of target genes. TaqMan analysis of paired tumor/normal samples revealed 1.5- to 2.6-fold overexpression of polymorphic miR-146a* in 7 of 8 tumors compared with the unaffected part of the same gland. The microarray data showed that widely different transcriptomes occurred in the tumors and in unaffected parts of the thyroid from GC and GG patients. The modulated genes are mainly involved in regulation of apoptosis leading to exaggerated DNA-damage response in heterozygotes potentially explaining the predisposition to cancer. We propose that contrary to previously held views transcripts from the passenger strand of miRs can profoundly affect the downstream effects. Heterozygosity for polymorphisms within the premiR sequence can cause epistasis through the production of additional mature miRs. We propose that mature miRs from the passenger strand may regulate many genetic processes.

Although papillary thyroid carcinoma (PTC) displays strong heritability, no predisposing germ-line mutations have been found. We show that a common G/C polymorphism (rs2910164) within the pre-miR-146a sequence reduced the amount of pre- and mature miR-146a from the C allele 1.9- and 1.8-fold, respectively, compared with the G allele. This is matched by a similar decrease in the amount of each pre-miR generated from the corresponding pri-miR-146a in an in vitro processing reaction. The C allele also interfered with the binding of a nuclear factor to pre-miR-146a. The reduction in miR-146a led to less efficient inhibition of target genes involved in the Toll-like receptor and cytokine signaling pathway (TRAF6, IRAK1), and PTC1 (also known as CCDC6 or H4), a gene frequently rearranged with RET proto-oncogene in PTC. In an association study of 608 PTC patients and 901 controls, we found marked differences in genotype distribution of rs2910164 (P = 0.000002), the GC heterozygous state being associated with an increased risk of acquiring PTC (odds ratio = 1.62, P = 0.000007), and both homozygous states protective with odds ratio = 0.42 for the CC genotype (P = 0.003) and odds ratio = 0.69 for the GG genotype (P = 0.0006). Moreover, 4.7% of tumors had undergone somatic mutations of the SNP sequence. Thus, our data suggest that a common polymorphism in pre-miR-146a affects the miR expression, contributes to the genetic predisposition to PTC, and plays a role in the tumorigenesis through somatic mutation. Preliminary evidence suggests that these effects are mediated through target genes whose expression is affected by the SNP status.

Context: IL-23 and its receptor (IL-23R) guide T cells toward the T-helper 17 phenotype. IL-23R single nucleotide polymorphisms (SNPs) have been associated with several autoimmune diseases, including Crohn's disease and rheumatoid arthritis., Objective: Our objective was to determine whether variants in the IL-23R gene are associated with Graves' disease (GD) and Graves' ophthalmopathy (GO)., Design and Participants: A total of 216 North American Caucasian GD patients and 368 healthy controls were genotyped for four SNPs spanning the IL-23R gene. SNPs rs11209026 and rs7530511 were genotyped using the TaqMan allelic discrimination assays (Applied Biosystems, Foster City, CA), and SNPs rs2201841 and rs10889677 were genotyped using a fluorescent-based restriction fragment length polymorphism method., Results: The A allele of rs2201841 was present in 78.8% of GD patients with GO and 64.7% of controls [P=1.1x10(-4); odds ratio (OR)=2.04]; the AA genotype was also significantly increased in GO patients compared with controls (62.5 and 41%, respectively; P=1.0x10(-4); OR=2.4). The C allele of rs10889677 was present in 78.6% of GO patients and 64.5% of controls (P=1.3x10(-4); OR=2.03), and the CC genotype was also significantly increased in GO patients vs. controls (62.1 and 41.0%, respectively; P=1.4x10(-4); OR=2.36). The TT genotype of rs7530511 was significantly associated with GD, but not specifically with GO; it was present in 2.5% of GD patients and 0.3% of controls (P=0.02; OR=9.4). The rs11209026 SNP, which is the most strongly associated with Crohn's disease, was not associated with GD or GO in our data set., Conclusions: Variants in the IL-23R gene are strongly associated with GO. These variants may predispose to GO by changing the expression and/or function of IL-23R, thereby promoting a proinflammatory signaling cascade.

Objective. Smoking, a major risk factor for cardiovascular morbidity and mortality, may be particularly harmful to women. Sympathetic and hemodynamic responses to cigarette smoking may be implicated in the link between smoking and acute cardiovascular events. We tested the hypothesis that acute effects of smoking on cardiovascular function are potentiated in women compared with men. Methods. We examined the effects of cigarette smoking and sham smoking on muscle sympathetic nerve activity, blood pressure and heart rate in 20 female and 20 male middle-aged healthy habitual smokers. Results. Sham smoking had no effect on muscle sympathetic nerve activity, blood pressure, or heart rate. Although cigarette smoking increased average systolic blood pressure and heart rate in both females and males, systolic blood pressure increased more in women (12+/-2 mmHg) than in men (6+/-2 mmHg; p = 0.02), as did heart rate (16+/-2 beats/min in women vs 9+/-2 beats/min in men; p = 0.002). Female smokers also had greater smoking-related increases in systolic blood pressure variability compared with males (2.2+/-0.6 vs 0.4+/-0.4 mmHg, respectively; p = 0.01) and greater decreases in RR variability (-28+/-5 vs -7+/-4 ms; p = 0.002). Despite the potentiated blood pressure increase in females, which would be expected to inhibit sympathetic activity to a greater extent in females than in males, changes in muscle sympathetic nerve activity during smoking were similar in both sexes. Conclusions. Acute pressor and tachycardic effects of smoking are potentiated in women compared with men. These findings may have important implications for understanding increased vulnerability to acute cardiovascular events in women who smoke.

Graves disease (GD) is an autoimmune disorder with genetic predisposition. The polymorphisms 47A-->G (Arg16Gly) and 79C-->G (Gln27Glu) of the adrenergic beta-2 receptor (ADRB2) gene in the 5q32 region affect the functional reaction to adrenergic stimulation, which contributes to the regulation of immunological response. The -367T-->C polymorphism within the 5'-leading regulatory sequence affects ADRB2 transcriptional activity. The aim of the present study was to investigate whether ADRB2 gene variants are associated with susceptibility to GD. All polymorphisms were studied in Polish GD patients (n=300) and healthy control subjects without a family history of autoimmune disorders (n=301). Genotypes were determined by the MassARRAY system (Sequenom, San Diego, CA). Gly16 and Gln27 allele frequencies were 61.4% and 55.2% among healthy controls, almost the same as previously reported in 4441 white participants of a cardiovascular health study. We found a higher risk of GD in Gln27 carriers (CC or CG genotypes) than in Glu27 homozygous (GG genotype) participants (OR=1.99, 95% CI: 1.27-3.12, p=0.003, pcorr=0.03). The frequency of the 79GG protective genotype was significantly smaller in the GD patients without symptoms of Graves ophthalmopathy compared to controls (10% vs 22%, OR=0.41, 95% CI: 0.234-0.706, p=0.0017, pcorr=0.015). We didn't find any association of -367T--> or 47A-->G genotypes/alleles with Graves disease, however, haplotype analysis has shown a significant difference in haplotype distribution between patients and controls (the global p=0.001) with increased -367T/47A/79C haplotype frequency in GD patients compared to controls (34% vs 25%, p=0.00073, pcorr=0.0044). In conclusion, Gln27 carriers (79CC or 79GC genotypes) have increased risk of Graves disease. Our results suggest that ADRB2 plays a role in susceptibility to Graves disease in humans.

Apart from alterations in the RET/PTC-RAS-BRAF pathway, comparatively little is known about the genetics of papillary thyroid carcinoma (PTC). We show that numerous microRNAs (miRNAs) are transcriptionally up-regulated in PTC tumors compared with unaffected thyroid tissue. A set of five miRNAs, including the three most up-regulated ones (miR-221, -222, and -146), distinguished unequivocally between PTC and normal thyroid. Additionally, miR-221 was up-regulated in unaffected thyroid tissue in several PTC patients, presumably an early event in carcinogenesis. Tumors in which the up-regulation (11- to 19-fold) of miR-221, -222, and -146 was strongest showed dramatic loss of KIT transcript and Kit protein. In 5 of 10 such cases, this down expression was associated with germline single-nucleotide changes in the two recognition sequences in KIT for these miRNAs. We conclude that up-regulation of several miRs and regulation of KIT are involved in PTC pathogenesis, and that sequence changes in genes targeted by miRNAs can contribute to their regulation.

Sialylation of cell components is an important immunomodulating mechanism affecting cell response to hormones and adhesion molecules. To study alterations in sialic acid metabolism in Graves' disease (GD) we measured the following parameters in various human thyroid tissues: lipid-bound sialic acid (LBSA) content, ganglioside profile, total sialyltransferase activity, and the two major sialyltransferase mRNAs for sialyltransferase-1 (ST6Gal I) and for sialyltransferase-4A (ST3Gal I). Fragments of toxic thyroid nodules (TN), nontoxic thyroid nodules (NN) and nontumorous tissue from patients with nodular goiter or thyroid cancer were used as a control (C). The LBSA content and sialyltransferase activity were the highest in the GD group (164 +/- 4.44 versus 120 +/- 2.00 nmoL/g, p = 0.005 and 1625 +/- 283.5 versus 324 +/- 54.2 cpm/mg of protein, p < 0.005 compared to control group C). Ganglioside profile in the GD group was similar to that in control tissues. Sialyltransferase- 1 mRNA and sialyltransferase-4A mRNA levels were significantly higher in the GD group than in the control group (12.52 +/- 6.90 versus 2.54 +/- 1.24 arbitrary units, p < 0.005 and 2,49 +/- 1.16 versus 1.23 +/- 0.46 arbitrary units, p < 0.05, respectively). There was a positive correlation between the increased sialyltransferase-1 mRNA level and the TSH-receptor antibody titer determined by the TRAK test. These results indicate that sialyltransferases expression and activity are increased in GD. Exact mechanism of this upregulation remains unknown, though one of possible explanations is the activation of the thyrotropin (TSH) receptor.

Tumor necrosis factor (TNF)-alpha plays a central role in the development of ophthalmopathy in patients with Graves' disease (GD). The aim of this study was to investigate the association of TNF promoter polymorphisms at positions -1031 (T-1031C), -863 (C-863A), -857 (C-857T), -308 (G-308A), and -238 (G-238A) with Graves' ophthalmopathy (GO). We studied the distribution of TNF and human leukocyte antigen (HLA) DRB1 alleles in 228 Polish white patients with GD, 106 of whom had ophthalmopathy (NOSPECS class > or = III) and 248 healthy subjects. TNF -308A and HLA-DRB1*03 alleles were significantly increased in patients with GD compared with healthy subjects. Stratification analysis revealed no independent association of -308A with GD when the DRB1*03 status was considered. Subdividing GD according to eye involvement revealed that the distribution of TNF promoter haplotypes differed significantly in patients with or without ophthalmopathy. The haplotype containing the -238A allele was absent in GO. The association of G-238A with GO was independent of DRB1 alleles. These results indicate that TNF G-308A is associated with susceptibility to GD (however, this association is not independent of HLA-DRB1*03) and that TNF G-238A is associated with the development of ophthalmopathy, suggesting that G-238A or a gene in linkage disequilibrium may be disease modifying in GD.

Objective: In patients with Graves' disease (GD), an elevation of serum immunoglobulin E (IgE) has been recently reported to be associated with the severity of hyperthyroidism and ophthalmopathy. Interleukin 13 (IL-13) is a major cytokine involved in IgE synthesis and therefore may be a potential candidate gene contributing to the development of GD or influencing the clinical course of the disease., Design: In a case-control study, we examined IL-13 gene single-nucleotide polymorphisms in the 5' promoter region at position -1112 (C to T change, termed as C-1112T) and in exon 4 at position 2044 (G to A change, G2044A, which results in an amino acid exchange Arg130Gln) in 261 patients with GD. The control groups consisted of healthy young subjects (n=168) and subjects over 100 years old with no history of autoimmune or allergic diseases recruited from the Polish Centenarians Project (n=50)., Measurements: C-1112T and G2044A polymorphisms were defined by fluorescent single-strand conformational polymorphism and by restriction fragment length polymorphism analysis, respectively., Results: In patients with GD, the distribution of IL-13 alleles (-1112T 31%; 2044A 25%) and genotypes (-1112T/T 10%; 2044A/A 7%) did not differ significantly compared to control groups. Subdividing GD patients according to clinically evident ophthalmopathy (NOSPECS class III or higher, n=93) revealed no significant differences in the frequencies of -1112T allele (33%vs. 29%; P=0.4), -1112T/T genotype (13%vs. 8%; P=0.3), 2044A allele (27%vs. 24%; P=0.5) and 2044A/A genotype (9%vs. 7%; P=0.7) between GD patients with and without eye involvement. In order to analyse the association with the severity of hyperthyroidism, we examined patients with a first onset of GD treated with antithyroid drugs (n=32). IL-13 genotypes were not associated with the laboratory findings at diagnosis (thyroid volume, serum levels of FT4, TRAb, TPOAb, TGAb) and with the outcome of antithyroid drug treatment., Conclusions: Our results suggest that IL-13 gene polymorphisms at positions -1112 (C-->T) and 2044 (G-->A): (1) do not confer genetic susceptibility to Graves' disease; (2) do not contribute to the development of clinically evident ophthalmopathy; (3) are not associated with severity of hyperthyroidism.