Skip to main content
  • Research article
  • Open access
  • Published:

Decrease in thyroid adenoma associated (THADA) expression is a marker of dedifferentiation of thyroid tissue

Abstract

Background

Thyroid adenoma associated (THADA) has been identified as the target gene affected by chromosome 2p21 translocations in thyroid adenomas, but the role of THADA in the thyroid is still elusive. The aim of this study was to quantify THADA gene expression in normal tissues and in thyroid hyper- and neoplasias, using real-time PCR.

Methods

For the analysis THADA and 18S rRNA gene expression assays were performed on 34 normal tissue samples, including thyroid, salivary gland, heart, endometrium, myometrium, lung, blood, and adipose tissue as well as on 85 thyroid hyper- and neoplasias, including three adenomas with a 2p21 translocation. In addition, NIS (sodium-iodide symporter) gene expression was measured on 34 of the pathological thyroid samples.

Results

Results illustrated that THADA expression in normal thyroid tissue was significantly higher (p < 0.0001, exact Wilcoxon test) than in the other tissues. Significant differences were also found between non-malignant pathological thyroid samples (goiters and adenomas) and malignant tumors (p < 0.001, Wilcoxon test, t approximation), anaplastic carcinomas (ATCs) and all other samples and also between ATCs and all other malignant tumors (p < 0.05, Wilcoxon test, t approximation). Furthermore, in thyroid tumors THADA mRNA expression was found to be inversely correlated with HMGA2 mRNA. HMGA2 expression was recently identified as a marker revealing malignant transformation of thyroid follicular tumors. A correlation between THADA and NIS has also been found in thyroid normal tissue and malignant tumors.

Conclusions

The results suggest THADA being a marker of dedifferentiation of thyroid tissue.

Peer Review reports

Background

Benign thyroid tumors and hyperplasias of follicular epithelial origin belong to the cytogenetically best analyzed human epithelial tumors.

Cytogenetic aberrations have been detected in approximately 20% of these lesions [1]. Translocations of chromosomal band 2p21 are the second most frequent structural chromosomal rearrangement, representing a particular cytogenetic subgroup [2]. The target gene has been identified and referred to as thyroid adenoma associated (THADA) [3].

The full length cDNA of THADA consists of 6,134 bp distributed over 38 exons [GenBank: NM_022065]. There are two splice-variants, one lacking exons 27 and 28 [3], and the other without exons 16 and 17. The THADA protein has three isoforms corresponding to the three different transcript variants with 1953 [GenBank: NP_071348], 1879, and 1832 amino acids, respectively. In adenomas with 2p21 translocations Rippe et al. found different types of fusion variants of THADA[3]. In each case, THADA was truncated after exon 28 and ectopic sequences fused to it were not correlated to any known gene. Thus, it has been speculated that the truncation rather than the fusion to ectopic coding sequences is the critical event for the development of the tumor [3].

Studies by Drieschner et al. [4] revealed that the mRNA, the protein size, and the genomic organization is conserved among Homo sapiens, Canis familiaris, Chlorocebus aethiops, Gallus gallus, and Mus musculus. THADA proteins from the analyzed organisms showed significant assignments to the superfamily ARM repeat (SSF48371; Hidden Markov Models Superfamily database), indicating the presence of a protein-protein-interaction-domain of that type.

The exact function of THADA still remains unclear. Hypothetically, it belongs to the death receptor-interacting proteins and is assumed to bind to death receptor DR5 (Puduvalli VK and Ridgway L, GenBank accession reference note), involving it in the TRAIL-induced apoptosis. The truncated THADA derived from the rearranged allele might compete with the gene product of the normal allele thereby disturbing normal apoptosis of follicular cells, and subsequently altering the steady state between proliferation and cellular death leading to adenomatous growth in benign thyroid tumors with 2p21 translocations [3]. Nevertheless, there is a need for further studies elucidating the role of THADA in normal thyroid development and in tumorigenesis.

Recently, a THADA variant has also been linked to type 2 diabetes (T2D) [5], but this association has not been confirmed by the majority of further studies [620]. During a meta-analysis of three genome-wide association studies with individuals of European descent Zeggini et al. found evidence for an association of a SNP (rs7578597) in exon 24 of THADA and the susceptibility for T2D [5]. Further indication for a correlation between THADA and T2D was presented in several other publications [11, 14, 16, 17, 19], one reported an altered expression of THADA in pancreatic islets, using data from the Diabetes Genome Anatomy Project (DGAP) database [11]. In other investigations no correlation was detected [68, 10, 12, 13, 15, 18, 20], except for one publication [9], which reported an association between THADA SNP rs7578597 and a 2-h insulin level during an oral glucose tolerance test but no significant association between the THADA SNP and T2D risk, rendering the association disputable.

The aim of this study was to analyze THADA expression in thyroid tissue in comparison to other tissues and to thyroid hyper- and neoplasias to elucidate the possible correlation of THADA mRNA with thyroid differentiation and neoplastic growth.

Methods

Tissue specimen and RNA isolation

RNA from snap-frozen tissues was isolated using the RNeasy Mini Kit and RNeasy Lipid Tissue Mini Kit for the adipose tissue samples, respectively (QIAGEN, Hilden, Germany).

For the formalin-fixed paraffin-embedded (FFPE) tissues of thyroid tumors, histopathologic diagnoses were performed according to the World Health Organization Classification of Tumours [21] (table 1). As to RNA isolation, FFPE blocks were cut into six sections of 5 μm for each sample using a microtome. Total RNA isolations were performed using the Roche High Pure RNA Paraffin Kit (Roche, Mannheim, Germany) for the THADA expression investigation and the RNeasy FFPE Kit (QIAGEN, Hilden, Germany) for the NIS expression analysis. Three samples were cytogenetically characterized by 2p21 translocations. In all three cases, two of which published previously [22, 23], the breakpoints were narrowed down to the THADA locus. One of the anaplastic thyroid samples served as the source of a newly established cell line. Cytogenetical analysis revealed a highly complex karyotype with a range of 80 to 117 chromosomes (100.8 on average). Several marker chromosomes, telomeric associations, and double minutes were detected.

Table 1 Histology of the malignant thyroid lesions.

cDNA-synthesis and real-time PCR expression analysis

RNAs were reverse-transcribed into cDNA by M-MLV Reverse Transcriptase (Invitrogen, Karlsruhe, Germany). Real-time PCR was performed using the Applied Biosystems 7300 sequence detection system according to TaqMan Gene Expression Assay Protocol (Applied Biosystems, Darmstadt, Germany) in 96-well microtiter plates with a total volume of 20 μl. In case of TaqMan gene expression assay of THADA (assay number Hs00152982, Applied Biosystems, Foster City, USA), targeting exons 31-32, and of NIS (assay number Hs00166567_m1), each reaction consisted of 2 μl of cDNA reverse transcribed from 25 ng of total RNA, 10 μl of TaqMan Universal PCR Master Mix (Applied Biosystems), 1 μl of TaqMan assay and 7 μl of ddH2O. For the 18S rRNA assay, using 18S forward and 18S rev_1 primers [24], each reaction consisted of 2 μl of cDNA (1:10 diluted, with regard to higher expression of 18S rRNA) reverse transcribed from 25 ng of total RNA, 10 μl of TaqMan Universal PCR Master Mix, 600 nM of forward and reverse primers, 200 nM of 18S probe [24] and 5.4 μl of ddH2O.

Thermal cycling conditions were 2 min at 50°C followed by 10 min at 95°C, 50 cycles at 95°C for 15 s and 60°C for 1 min. A non-template control of amplification and two previous negative controls of cDNA synthesis (one without RNA and one missing Reverse Transcriptase) were included in each plate. Software Sequence Detection Software 1.2.3 (Applied Biosystems) was programmed with the reaction condition. All testing reactions were performed in triplicate.

Serial dilutions were made using cDNA derived from 25, 5, 1, 0.2, and 0.04 ng of total RNA from FFPE tissue of one thyroid adenoma for THADA and 18S rRNA, and from fresh frozen tissue of one normal thyroid sample for NIS. In each dilution, THADA, NIS, and 18S rRNA gene expression assays were performed using absolute quantification. Afterwards, the standard curves for both assays were plotted with the log ng of input cDNA for each dilution on the x-axis, and the matched CT value on the y-axis. Furthermore, in order to evaluate the differences of amplification efficiencies, the difference of two curve slopes was calculated. If the absolute difference of the slopes is less than 0.1, the amplification efficiencies of two assays are considered to be equal and the comparative CT method is valid (User Bulletin No. 2, ABI PRISM 7700 Sequence Detection System, Applied Biosystems). 18S rRNA was used as endogenous control as suggested previously [2528]. The 18S rRNA assay showed an amplification efficiency of 92.6% (slope = -3.514, R2 = 0.995). The THADA assay had an amplification efficiency of 92.0% (slope = -3.531) and an R2-value of 0.96. For NIS, the amplification efficiency was 93.4% (slope = -3.4917), the coefficient of determination amounted to 0.997). As recommended for FFPE samples [24, 2931] the fragment sizes amplified by all three assays were small, ranging between 60 and 78 bp, a validation of these values was performed via gelelectrophoresis of the PCR-products (data not shown). When applying the comparative CT method, one histological normal thyroid tissue was used as calibrator sample. Afterwards, data were compared with results from conventional histology.

For statistical analysis, the Wilcoxon signed rank test was used to compare average values (two-sided, exact version for at most 40 cases involved, otherwise using the t approximation); relationships were quantified by linear regression and Spearman's rank correlation coefficient. Sensitivity, specificity and decision limits were calculated from non-parametric density estimations. Therefore, sensitivity and specificity may differ from raw empirical values and decision limits need not coincide with measured values. A p-value of less than 0.05 was considered significant.

Ethics Statement

The use of human thyroid samples for this study was approved by the local medical ethics committee (Ethikkommission bei der Ärztekammer Bremen) and followed the guidelines of the declaration of Helsinki. Only samples that were initially taken for diagnostic purposes were secondarily used for the present study. During pathological examination, a sample of the tissue was snap-frozen. The procedure was approved by the local ethics committee. Because the samples were deidentified and were considered as samples normally discarded, the committee felt that there was no specific patient consent necessary.

As for the normal tissue samples, these were anonymously collected for earlier studies, each following the guidelines of the declaration of Helsinki.

Results

THADA expression in normal tissues

Thirty-four snap-frozen samples from eight different tissues were tested for the level of THADA expression.

The mean level per tissue type ranged from 1 (blood) to 6.14 (thyroid), and the lowest single value for a thyroid sample (4.04) was above the highest one (3.39, myometrium) from any of the other tissues (Figure 1). Accordingly, statistical analysis using Wilcoxon's exact signed rank showed significant differences between normal thyroid tissues and the group of all other tissues (p < 0.0001). Using the THADA expression to discriminate between thyroid and non-thyroid tissue, a sensitivity of 82.5%, a specificity of 97.4% and an efficiency of 95.2% with a decision limit value of 4.23 were achieved.

Figure 1
figure 1

THADA expression in normal tissues (snap-frozen samples). Boxplots for the relative quantifications of THADA gene expression in normal tissues; tissue type at x-axis. (*): p < 0.0001 compared to all other tissues jointly (exact Wilcoxon signed rank test). Boxes contain the inner 50% of all values and a bar at the position of the median, whiskers extend to the extrema of values or to 1.5 * box height, whichever is smaller. The plus sign shows the arithmetic mean. (n: number of samples).

THADA expression in thyroid tumors

Ninety-three formalin-fixed-paraffin-embedded thyroid samples, including eight normal tissues (from four patients), 18 goiters, 35 benign, and 32 malignant tumors were measured. For single tumor samples the expression ranged between 0.065 (anaplastic carcinoma) and 2.986 (follicular adenoma) in relation to normal tissue, i.e. a ratio of 1 : 45.94. Samples with a 2p21 translocation showed a level of expression of 1.123, 1.624, and 0.662 fold, respectively. The mean values for the different tumor entities ranged from 0.423 (anaplastic carcinoma) to 1.156 (adenoma) (Figure 2 and table 2).

Figure 2
figure 2

THADA expression in thyroid hyper- and neoplasias (FFPE samples). Boxplots for the relative quantifications of THADA gene expression in thyroid normal tissue, goiter, benign and malignant tumors; normal tissue and hyper-/neoplasia type at x-axis. Boxes contain the inner 50% of all values and a bar at the position of the median, whiskers extend to the extrema of values or to 1.5 * box height, whichever is smaller, isolated symbols indicate values outside this range. The plus sign shows the arithmetic mean. (n: number of samples).

Table 2 Detailed view of THADA expression in thyroid hyper- and neoplasias

Significant differences of THADA expression were noted between benign and malignant thyroid lesions. Wilcoxon's signed rank test showed a highly significant difference comparing the joint group of goiters and benign tumors with malignant tumors (p = 0.0009).

Using the exact Wilcoxon test, no significant differences were detected comparing the level of THADA expression between normal tissue and benign lesions (p = 0.2802) and papillary carcinomas (p = 0.2170). In contrast, significant differences were found between anaplastic carcinomas (ATCs), the most dedifferentiated type of thyroid tumors, and all other samples (p = 0.0107) and ATCs and all other malignant tumors (p = 0.0234). Comparing anaplastic carcinomas with each single group, the difference in expression between ATCs and goiters (p = 0.0049) and adenomas (p = 0.0058) were marked as significant. As this finding was a result of systematically comparing anaplastic carcinomas with the other lesions, a Bonferroni correction for multiple testing was used (corrected α = 0.0083). Without the need of correcting for multiple testing also normal tissue and papillary carcinoma would have been assessed as significantly different from anaplastic carcinoma (p = 0.0485 and p = 0.0350, respectively). Overall, significant results were mostly seen with the group of anaplastic carcinomas, indicating a relative stable level of expression in comparatively differentiated tissues with a significant reduction only in dedifferentiated tissues.

Recently HMGA2 expression has been shown to indicate thyroid malignancy and can thus be considered marking the dedifferentiation of thyroid epithelium [3234]. As to the study by Belge et al. [32] and the present one 48 samples were identical in both studies (seven normal tissues, one goiter, 15 adenomas and 25 carcinomas, including three anaplastic carcinomas). For these, RNA was isolated from adjacent cuts of the same FFPE block and, except for the different qRT-PCR assays, all samples were treated identical in both investigations. Thus, it was feasible to check these samples for a possible correlation between THADA and HMGA2. Using Spearman's rank correlation, there was a highly significant inverse correlation between THADA and HMGA2 expression (correlation coefficient = -0.452; p = 0.0015), further underlining a possible role of THADA in thyroid differentiation.

NIS (sodium-iodide symporter), the transmembrane glycoprotein accountable for the uptake of iodine in thyroid cells, was found to be a marker of thyroid differentiation [3538]. To validate our findings NIS expression was measured in 41 samples, including seven normal tissue samples, six nodular goiters, five adenomas, and 23 carcinomas (15 papillary, four follicular, and all four anaplastic thyroid carcinomas). Using Spearman's rank correlation, no significant correlation (p = 0.1288) was detected comparing THADA and NIS expression from all samples. By contrast, a significant correlation was found constraining the analysis to the follicular and papillary carcinoma samples (p = 0.0497, r = 0.456, n = 19), an even stronger correlation between the expression of THADA and NIS was found in normal and all malignant samples (p = 0.0021, r = 0.540, n = 30), and in normal tissue and anaplastic carcinomas (p = 0.0128, r = 0.718, n = 11)

Transcription factors binding to THADA

Using the SABiosciene DECODE Transcription Factor Search, no THADA-promotor binding sites for thyroid-specific transcription factors paired box gene 8 (pax8), thyroid transcription factor 1 (TTF1), also known as NK2 homeobox 1 (NKX2-1), and thyroid transcription factor (TTF-2), sometimes referred to as forkhead box protein E1 (FOXE1), were found. Amongst others cAMP response element-binding protein (CREB), activating transcription factor (ATF-2), c-Jun, hepatic leukemia factor (Hlf), and germ cell nuclear factor (GCNF) were marked as relevant, FOXC1, Nkx2-2, Nkx2-5, and Nkx6-1 were displayed with low relevance (data not shown). HHEX (hematopoietically expressed homeobox) has been found to be expressed in the adult thyroid gland and in differentiated thyroid cell lines and to be correlated with thyroid differentiation [3941], but is not included in the SABiosciene DECODE Transcription Factor Search. A manual search for this transcription factor revealed no assured binding sites in the THADA promoter.

Discussion

In this study, THADA turned out to be highly expressed in the thyroid compared to other normal tissues. In a group of eight different types of tissue thyroid samples showed a significantly higher THADA mRNA expression than salivary gland, lung, heart, myometrium, endometrium, blood, and adipose tissue, hinting at a possibly important role of THADA in the thyroid.

The results in part contradict data available online. NCBI ESTProfileViewer predicted a higher expression in heart and lung tissue and a slightly lower in the thyroid. For uterus and blood the data are in concordance with those obtained from the EST-based estimates. For salivary gland and adipose tissue the TPM (transcripts per million)-values are zero, this could be due to an overall small EST pool (20155 ESTs for salivary gland, 13106 ESTs for adipose tissue), resulting in less than one gene EST (all normal tissues average: 31073 ESTs per gene EST). Comparison to Affymetrix GeneChip Human Genome array-based results from The Genomics Institute of the Novartis Research Foundation (GNF) showed similar discrepancies. There are three probes, one (gnf1h10751_at) is diverging considerably from the other two and was therefore omitted. Compared to our data both remaining probes resulted in similarly average Spearman's rank correlation coefficients and no significances (p ≥ 0.2). GNF results showed thyroid as the tissue with the highest THADA expression but less distinct from the other tissues. Overall, the more precise and reliable qRT-PCR-method disclosed results that are diverging from those available from online databases.

Furthermore, evidence that THADA expression is associated to thyroid differentiation has been presented. Analysis of 93 thyroid FFPE samples revealed significant differences between benign and malignant thyroid lesions, especially when comparing the group of anaplastic carcinomas with other types of lesions. Despite one outlier with an expression level almost identical to normal tissue, the values were significantly lower compared to all other samples as well as to all other malignant tumors. A comparison of the expression level of THADA and NIS (sodium-iodide symporter) confirmed these observations. Amongst others, a significant correlation between THADA and this well established marker of thyroid differentiation [3538] has been detected in normal tissue and anaplastic carcinomas. This suggests that THADA expression decreases with dedifferentiation of the thyroid epithelium. This hypothesis is further supported by the significant inverse correlation between the expression of THADA and HMGA2. Belge et al. [32] showed that HMGA2 is significantly overexpressed in malignant thyroid tumors compared to benign lesions. As a rule, a high HMGA2 expression seems to be accompanied by a low THADA expression. As yet the underlying mechanism is unknown but it does not seem to involve thyroid-specific transcription factors, since no binding sites for pax8, TTF-1 and -2 were found. However, the SABiosciene DECODE Transcription Factor Search revealed a binding site of the cAMP response element-binding protein (CREB). CREB has been shown to regulate diverse cellular responses, including differentiation [42], targeted expression of dominant-negative mutants of CREB in transgenic mice has been associated with thyroid hypoplasia [43]. cAMP indirectly plays a crucial role in the differentiation of endocrine tissues [43], including the thyroid [44, 45]. Thus one might speculate about an involvement in the decreased expression of THADA in dedifferentiated thyroid cells.

In thyroid adenomas THADA was frequently found to be truncated [3]. Whereas the intact THADA may be involved in maintaining the differentiation of thyroid epithelium, the truncated allele might play a key role in tumor development of the thyroid. While competing with the full-length protein translated from the normal allele of THADA the altered protein derived from the truncated gene might lead to an impaired induction of apoptosis, and subsequently give rise to an increased cell proliferation leading to benign thyroid tumors with 2p21 translocations [3], without significant changes of the expression level.

Conclusions

THADA expression, though not restricted to the follicular cells of the thyroid, is higher in the thyroid than in other tissues tested (salivary gland, heart, endometrium, myometrium, lung, blood, and adipose tissue). As to its normal function, THADA expression has been found to be decreased in anaplastic carcinomas and to be correlated with the expression of NIS, a marker of thyroid differentiation, and inversely correlated with that of HMGA2, a marker of malignant transformation of the thyroid and cancer stemness. It may thus have essential functions in maintaining the differentiation of the follicular epithelium.

References

  1. Belge G, Roque L, Soares J, Bruckmann S, Thode B, Fonseca E, Clode A, Bartnitzke S, Castedo S, Bullerdiek J: Cytogenetic investigations of 340 thyroid hyperplasias and adenomas revealing correlations between cytogenetic findings and histology. Cancer Genet Cytogenet. 1998, 101: 42-48. 10.1016/S0165-4608(97)00057-5.

    Article  CAS  PubMed  Google Scholar 

  2. Bol S, Belge G, Thode B, Bartnitzke S, Bullerdiek J: Structural abnormalities of chromosome 2 in benign thyroid tumors. Three new cases and review of the literature. Cancer Genet Cytogenet. 1999, 114: 75-77. 10.1016/S0165-4608(99)00028-X.

    Article  CAS  PubMed  Google Scholar 

  3. Rippe V, Drieschner N, Meiboom M, Murua Escobar H, Bonk U, Belge G, Bullerdiek J: Identification of a gene rearranged by 2p21 aberrations in thyroid adenomas. Oncogene. 2003, 22: 6111-6114. 10.1038/sj.onc.1206867.

    Article  CAS  PubMed  Google Scholar 

  4. Drieschner N, Kerschling S, Soller JT, Rippe V, Belge G, Bullerdiek J, Nimzyk R: A domain of the thyroid adenoma associated gene (THADA) conserved in vertebrates becomes destroyed by chromosomal rearrangements observed in thyroid adenomas. Gene. 2007, 403: 110-117. 10.1016/j.gene.2007.06.029.

    Article  CAS  PubMed  Google Scholar 

  5. Zeggini E, Scott LJ, Saxena R, Voight BF, Marchini JL, Hu T, de Bakker PI, Abecasis GR, Almgren P, Andersen G, Ardlie K, Boström KB, Bergman RN, Bonnycastle LL, Borch-Johnsen K, Burtt NP, Chen H, Chines PS, Daly MJ, Deodhar P, Ding CJ, Doney AS, Duren WL, Elliott KS, Erdos MR, Frayling TM, Freathy RM, Gianniny L, Grallert H, Grarup N, Groves CJ, Guiducci C, Hansen T, Herder C, Hitman GA, Hughes TE, Isomaa B, Jackson AU, Jørgensen T, Kong A, Kubalanza K, Kuruvilla FG, Kuusisto J, Langenberg C, Lango H, Lauritzen T, Li Y, Lindgren CM, Lyssenko V, Marvelle AF, Meisinger C, Midthjell K, Mohlke KL, Morken MA, Morris AD, Narisu N, Nilsson P, Owen KR, Palmer CN, Payne F, Perry JR, Pettersen E, Platou C, Prokopenko I, Qi L, Qin L, Rayner NW, Rees M, Roix JJ, Sandbaek A, Shields B, Sjögren M, Steinthorsdottir V, Stringham HM, Swift AJ, Thorleifsson G, Thorsteinsdottir U, Timpson NJ, Tuomi T, Tuomilehto J, Walker M, Watanabe RM, Weedon MN, Willer CJ, Wellcome Trust Case Control Consortium, Illig T, Hveem K, Hu FB, Laakso M, Stefansson K, Pedersen O, Wareham NJ, Barroso I, Hattersley AT, Collins FS, Groop L, McCarthy MI, Boehnke M, Altshuler D: Meta-analysis of genome-wide association data and large-scale replication identifies additional susceptibility loci for type 2 diabetes. Nat Genet. 2008, 40: 638-645. 10.1038/ng.120.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Grarup N, Andersen G, Krarup NT, Albrechtsen A, Schmitz O, Jørgensen T, Borch-Johnsen K, Hansen T, Pedersen O: Association testing of novel type 2 diabetes risk-alleles in the JAZF1, CDC123/CAMK1D, TSPAN8, THADA, ADAMTS9, and NOTCH2 loci with insulin release, insulin sensitivity and obesity in a population-based sample of 4,516 glucose-tolerant middle-aged Danes. Diabetes. 2008, 57: 2534-2540. 10.2337/db08-0436.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Staiger H, Machicao F, Kantartzis K, Schäfer SA, Kirchhoff K, Guthoff M, Silbernagel G, Stefan N, Fritsche A, Häring HU: Novel meta-analysis-derived type 2 diabetes risk loci do not determine prediabetic phenotypes. PLoS One. 2008, 3: e3019-10.1371/journal.pone.0003019.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Boesgaard TW, Gjesing AP, Grarup N, Rutanen J, Jansson PA, Hribal ML, Sesti G, Fritsche A, Stefan N, Staiger H, Häring H, Smith U, Laakso M, Pedersen O, Hansen T, the EUGENE2 Consortium: Variant near ADAMTS9 known to associate with type 2 diabetes is related to insulin resistance in offspring of type 2 diabetes patients-EUGENE2 study. PLoS One. 2009, 4: e7236-10.1371/journal.pone.0007236.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Hu C, Zhang R, Wang C, Wang J, Ma X, Lu J, Qin W, Hou X, Wang C, Bao Y, Xiang K, Jia W: PPARG, KCNJ11, CDKAL1, CDKN2A-CDKN2B, IDE-KIF11-HHEX, IGF2BP2 and SLC30A8 are associated with type 2 diabetes in a Chinese population. PLoS One. 2009, 4: e7643-10.1371/journal.pone.0007643.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Kang ES, Kim MS, Kim CH, Nam CM, Han SJ, Hur KY, Ahn CW, Cha BS, Kim SI, Lee HC, Kim YS: Association of common type 2 diabetes risk gene variants and posttransplantation diabetes mellitus in renal allograft recipients in Korea. Transplantation. 2009, 88: 693-698. 10.1097/TP.0b013e3181b29c41.

    Article  CAS  PubMed  Google Scholar 

  11. Parikh H, Lyssenko V, Groop LC: Prioritizing genes for follow-up from genome wide association studies using information on gene expression in tissues relevant for type 2 diabetes mellitus. BMC Medical Genomics. 2009, 2: 72-10.1186/1755-8794-2-72.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Raj SM, Howson JMM, Walker NM, Cooper JD, Smyth DJ, Field SF, Stevens HE, Todd JA: No association of multiple type 2 diabetes loci with type 1 diabetes. Diabetologia. 2009, 52: 2109-2116. 10.1007/s00125-009-1391-y.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sanghera DK, Been L, Ortega L, Wander GS, Mehra NK, Aston CE, Mulvihill JJ, Ralhan S: Testing the association of novel meta-analysis-derived diabetes risk genes with type II diabetes and related metabolic traits in Asian Indian Sikhs. J Hum Genet. 2009, 54: 162-168. 10.1038/jhg.2009.7.

    Article  CAS  PubMed  Google Scholar 

  14. Schleinitz D, Tönjes A, Böttcher Y, Dietrich K, Enigk B, Koriath M, Scholz GH, Blüher M, Zeggini E, McCarthy MI, Kovacs P, Stumvoll M: Lack of significant effects of the type 2 diabetes susceptibility loci JAZF1, CDC123/CAMK1D, NOTCH2, ADAMTS9, THADA, and TSPAN8/LGR5 on diabetes and quantitative metabolic traits. Horm Metab Res. 2009, 42: 14-22.

    Article  PubMed  Google Scholar 

  15. Stancáková A, Kuulasmaa T, Paananen J, Jackson AU, Bonnycastle LL, Collins FS, Boehnke M, Kuusisto J, Laakso M: Association of 18 confirmed susceptibility loci for type 2 diabetes with indices of insulin release, proinsulin conversion, and insulin sensitivity in 5,327 non-diabetic Finnish men. Diabetes. 2009, 58: 2129-2136. 10.2337/db09-0117.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Simonis-Bik AM, Nijpels G, van Haeften TW, Houwing-Duistermaat JJ, Boomsma DI, Reiling E, van Hove EC, Diamant M, Kramer MH, Heine RJ, Maassen JA, Slagboom PE, Willemsen G, Dekker JM, Eekhoff EM, de Geus EJ, 't Hart LM: Gene variants in the novel type 2 diabetes loci CDC123/CAMK1D, THADA, ADAMTS9, BCL11A, and MTNR1B affect different aspects of pancreatic beta-cell function. Diabetes. 2010, 59: 293-301. 10.2337/db09-1048.

    Article  CAS  PubMed  Google Scholar 

  17. Stuebe AM, Lyon H, Herring AH, Ghosh J, Wise A, North KE, Siega-Riz AM: Obesity and diabetes genetic variants associated with gestational weight gain. Am J Obstet Gynecol. 2010, 203: 283-e1-17

    Article  PubMed  PubMed Central  Google Scholar 

  18. Zhao J, Bradfield JP, Zhang H, Annaiah K, Wang K, Kim CE, Glessner JT, Frackelton EC, Otieno FG, Doran J, Thomas KA, Garris M, Hou C, Chiavacci RM, Li M, Berkowitz RI, Hakonarson H, Grant SF: Examination of all type 2 diabetes GWAS loci reveals HHEX-IDE as a locus influencing pediatric BMI. Diabetes. 2010, 59: 751-755. 10.2337/db09-0972.

    Article  CAS  PubMed  Google Scholar 

  19. Klimentidis YC, Divers J, Casazza K, Beasley TM, Allison DB, Fernandez JR: Ancestry-informative markers on chromosomes 2, 8 and 15 are associated with insulin-related traits in a racially diverse sample of children. Hum Genomics. 2011, 5: 79-89.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Vangipurapu J, Stančáková A, Pihlajamäki J, Kuulasmaa TM, Kuulasmaa T, Paananen J, Kuusisto J, Ferrannini E, Laakso M: Association of indices of liver and adipocyte insulin resistance with 19 confirmed susceptibility loci for type 2 diabetes in 6,733 non-diabetic Finnish men. Diabetologia. 2011, 54: 563-571. 10.1007/s00125-010-1977-4.

    Article  CAS  PubMed  Google Scholar 

  21. DeLellis RA, Lloyd RV, Heitz PU, Eng C, (Eds.): World Health Organization Classification of Tumours. Pathology and Genetics of Endocrine Organs. 2004, Lyon: IARC Press

    Google Scholar 

  22. Bol S, Belge G, Rippe V, Bullerdiek J: Molecular cytogenetic investigations define a subgroup of thyroid adenomas with 2p21 breakpoints clustered to a region of less than 450 kb. Cytogenet Cell Genet. 2001, 95: 189-191. 10.1159/000059344.

    Article  CAS  PubMed  Google Scholar 

  23. Drieschner N, Belge G, Rippe V, Meiboom M, Loeschke S, Bullerdiek J: Evidence for a 3p25 breakpoint hot spot region in thyroid tumors of follicular origin. Thyroid. 2006, 16: 1091-1096. 10.1089/thy.2006.16.1091.

    Article  CAS  PubMed  Google Scholar 

  24. Antonov J, Goldstein DR, Oberli A, Baltzer A, Pirotta M, Fleischmann A, Altermatt HJ, Jaggi R: Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization. Lab Invest. 2005, 85: 1040-1050. 10.1038/labinvest.3700303.

    Article  CAS  PubMed  Google Scholar 

  25. Bidart JM, Lacroix L, Evain-Brion D, Caillou B, Lazar V, Frydman R, Bellet D, Filetti S, Schlumberger M: Expression of Na+/I- symporter and Pendred syndrome genes in trophoblast cells. J Clin Endocr Metab. 2000, 85: 4367-4372. 10.1210/jc.85.11.4367.

    CAS  PubMed  Google Scholar 

  26. Bas A, Forsberg G, Hammarström S, Hammarström ML: Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. Scand J Immunol. 2004, 59: 566-573. 10.1111/j.0300-9475.2004.01440.x.

    Article  CAS  PubMed  Google Scholar 

  27. Macluskey M, Baillie R, Morrow H, Schor SL, Schor AM: Extraction of RNA from archival tissues and measurement of thrombospondin-1 mRNA in normal, dysplastic, and malignant oral tissues. Br J Oral Maxillofac Surg. 2006, 44: 116-123. 10.1016/j.bjoms.2005.03.001.

    Article  CAS  PubMed  Google Scholar 

  28. Nordén MM, Larsson F, Tedelind S, Carlsson T, Lundh C, Forssell-Aronsson E, Nilsson M: Down-regulation of the sodium/iodide symporter explains 131I-induced thyroid stunning. Cancer Res. 2007, 67: 7512-7517. 10.1158/0008-5472.CAN-07-0823.

    Article  PubMed  Google Scholar 

  29. Godfrey TE, Kim SH, Chavira M, Ruff DW, Warren RS, Gray JW, Jensen RH: Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 59 nuclease quantitative reverse transcription-polymerase chain reaction. J Mol Diagn. 2000, 2: 84-91. 10.1016/S1525-1578(10)60621-6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lehmann U, Kreipe H: Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods. 2001, 25: 409-418. 10.1006/meth.2001.1263.

    Article  CAS  PubMed  Google Scholar 

  31. Specht K, Richter T, Müller U, Walch A, Werner M, Höfler H: Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue. Am J Pathol. 2001, 158: 419-429. 10.1016/S0002-9440(10)63985-5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Belge G, Meyer A, Klemke M, Burchardt K, Stern C, Wosniok W, Loeschke S, Bullerdiek J: Upregulation of HMGA2 in thyroid carcinomas: a novel molecular marker to distinguish between benign and malignant follicular neoplasias. Genes Chromosomes Cancer. 2008, 47: 56-63. 10.1002/gcc.20505.

    Article  CAS  PubMed  Google Scholar 

  33. Chiappetta G, Ferrarob A, Vuttarielloa E, Monacoa M, Galdieroa F, De Simonea V, Califanoa D, Pallanteb P, Bottia G, Pezzulloa L, Pierantonib GM, Santorob M, Fusco A: HMGA2 mRNA expression correlates with the malignant phenotype in human thyroid neoplasias. Eur J Cancer. 2008, 44: 1015-1021. 10.1016/j.ejca.2008.02.039.

    Article  CAS  PubMed  Google Scholar 

  34. Prasad NB, Somervell H, Tufano RP, Dackiw AP, Marohn MR, Califano JA, Wang Y, Westra WH, Clark DP, Umbricht CB, Libutti SK, Zeiger MA: Identification of genes differentially expressed in benign versus malignant thyroid tumors. Clin Cancer Res. 2008, 14: 3327-3337. 10.1158/1078-0432.CCR-07-4495.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Arturi F, Russo D, Schlumberger M, du Villard JA, Caillou B, Vigneri P, Wicker R, Chiefari E, Suarez HG, Filetti S: Iodide symporter gene expression in human thyroid tumors. J Clin Endocrinol Metab. 1998, 83: 2493-2496. 10.1210/jc.83.7.2493.

    CAS  PubMed  Google Scholar 

  36. Dohán O, De la Vieja A, Paroder V, Riedel C, Artani M, Reed M, Ginter CS, Carrasco N: The sodium/iodide Symporter (NIS): characterization, regulation, and medical significance. Endocr Rev. 2003, 24: 48-77. 10.1210/er.2001-0029.

    Article  PubMed  Google Scholar 

  37. Ward LS, Santarosa PL, Granja F, da Assumpção LV, Savoldi M, Goldman GH: Low expression of sodium iodide symporter identifies aggressive thyroid tumors. Cancer Lett. 2003, 200: 85-91. 10.1016/S0304-3835(03)00392-6.

    Article  CAS  PubMed  Google Scholar 

  38. Li W, Ain KB: Human sodium-iodide symporter (hNIS) gene expression is inhibited by a trans-active transcriptional repressor, NIS-repressor, containing PARP-1 in thyroid cancer cells. Endocr Relat Cancer. 2010, 17: 383-398. 10.1677/ERC-09-0156.

    Article  CAS  PubMed  Google Scholar 

  39. Pellizzari L, D'Elia A, Rustighi A, Manfioletti G, Tell G, Damante G: Expression and function of the homeodomain-containing protein Hex in thyroid cells. Nucleic Acids Res. 2000, 28: 2503-2511. 10.1093/nar/28.13.2503.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. D'Elia AV, Tell G, Russo D, Arturi F, Puglisi F, Manfioletti G, Gattei V, Mack DL, Cataldi P, Filetti S, Di Loreto C, Damante G: Expression and localization of the homeodomain-containing protein HEX in human thyroid tumors. J Clin Endocrinol Metab. 2002, 87: 1376-1383. 10.1210/jc.87.3.1376.

    Article  PubMed  Google Scholar 

  41. Puppin C, Presta I, D'Elia AV, Tell G, Arturi F, Russo D, Filetti S, Damante G: Functional interaction among thyroid-specific transcription factors: Pax8 regulates the activity of Hex promoter. Mol Cell Endocrinol. 2004, 214: 117-125. 10.1016/j.mce.2003.10.061.

    Article  CAS  PubMed  Google Scholar 

  42. Wen AY, Sakamoto KM, Miller LS: The role of the transcription factor CREB in immune function. J Immunol. 2010, 185: 6413-6419. 10.4049/jimmunol.1001829.

    Article  CAS  PubMed  Google Scholar 

  43. Rosenberg D, Groussin L, Jullian E, Perlemoine K, Bertagna X, Bertherat J: Role of the PKA-regulated transcription factor CREB in development and tumorigenesis of endocrine tissues. Ann N Y Acad Sci. 2002, 968: 65-74. 10.1111/j.1749-6632.2002.tb04327.x.

    Article  CAS  PubMed  Google Scholar 

  44. Brunetti A, Chiefari E, Filetti S, Russo D: The 3',5'-cyclic adenosine monophosphate response element binding protein (CREB) is functionally reduced in human toxic thyroid adenomas. Endocrinology. 2000, 141: 722-730. 10.1210/en.141.2.722.

    CAS  PubMed  Google Scholar 

  45. Nguyen LQ, Kopp P, Martinson F, Stanfield K, Roth SI, Jameson JL: A dominant negative CREB (cAMP response element-binding protein) isoform inhibits thyrocyte growth, thyroid-specific gene expression, differentiation, and function. Mol Endocrinol. 2000, 14: 1448-1461. 10.1210/me.14.9.1448.

    Article  CAS  PubMed  Google Scholar 

Pre-publication history

Download references

Acknowledgements and funding

We thank U. Bonk, D. Krisponeit, T. Löning, E. Vollmer, T. Goldmann, and C. Frantzen for kindly providing the samples used in this analysis. There was no external funding for this study.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jörn Bullerdiek.

Additional information

Declaration of competing interests

The authors declare that they have no competing interests.

Authors' contributions

LK conceived and designed the study, carried out the molecular genetic studies, took part in the statistical analysis and the search for transcription factors and drafted the manuscript. GB provided the study material (FFPE and part of the snap-frozen samples), and revised the manuscript. KB carried out the clinical workout and performed the pathological analysis. SL performed the pathological determination of the samples (verification). WW carried out the statistical analysis. XF took part in the molecular studies. RN took part in the search for transcription factors. SM provided the study material (part of the snap-frozen samples) and took part in the analysis and interpretation of the obtained data. ND provided background information of certain samples and took part in the analysis and interpretation of the obtained data. VR took part in the analysis and interpretation of the obtained data. JB conceived the study and participated in its design and coordination and helped to draft and revised the manuscript. All authors have read and approved the final manuscript

Authors’ original submitted files for images

Below are the links to the authors’ original submitted files for images.

Authors’ original file for figure 1

Authors’ original file for figure 2

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Kloth, L., Belge, G., Burchardt, K. et al. Decrease in thyroid adenoma associated (THADA) expression is a marker of dedifferentiation of thyroid tissue. BMC Clin Pathol 11, 13 (2011). https://doi.org/10.1186/1472-6890-11-13

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/1472-6890-11-13

Keywords