Molekularzytogenetische und molekulargenetische Untersuchungen an Schilddrüsentumoren

The focus of the present study was on the molecular-cytogenetic analysis of the main cytogenetic subgroups in follicular lesions of the thyroid. Quantitative molecular-cytogenetic analysis of benign thyroid lesions revealed that much greater importance is attached to the main cytogenetic subgroups, i.e. trisomy 7 and rearrangements of 2p21 or 19q13.4, than by conventional cytogenetic analysis alone. For 2p21-rearrangements it was possible to identify and to further characterize the target gene, THADA, involved in these rearrangements. Further analyses suggest that the truncation of THADA is in correlation with the proliferation of epithelial cells und the genesis of benign thyroid lesions. For 19q13.4 rearrangements the breakpoints were narrowed down to a region of about 150 kb which is in close proximity to two miRNA clusters, C19MC and mir-371-3, which both are activated as a result of the rearrangements. The molecular-cytogenetic analysis of benign as well as malign follicular thyroid neoplasias with respect to the translocation t(2;3)(q13;p25) and the PAX8/PPARγ fusion gene, respectively, revealed that this aberration occurs only in a small fraction of follicular adenomas. Molecular-cytogenetic characterization of the 3p25 breakpoint region reveals that rearrangements of the 3p25 region occur independently of the 2q13 region and the PAX8 gene, respectively. Thus this region is considered as a hot spot region in thyroid neoplasias with follicular origin. In conclusion the results of the present study point out the status of molecular cytogenetics with respect to the analysis of tumor-specific chromosomal aberrations in general and in particular to the relevance of these aberrations to the pathogenesis of benign follicular thyroid lesions

The Two Stem Cell MicroRNA Gene Clusters C19MC and miR-371-3 Are Activated by Specific Chromosomal Rearrangements in a Subgroup of Thyroid Adenomas

Thyroid adenomas are common benign human tumors with a high prevalence of about 5% of the adult population even in iodine sufficient areas. Rearrangements of chromosomal band 19q13.4 represent a frequent clonal cytogenetic deviation in these tumors making them the most frequent non-random chromosomal translocations in human epithelial tumors at all. Two microRNA (miRNA) gene clusters i.e. C19MC and miR-371-3 are located in close proximity to the breakpoint region of these chromosomal rearrangements and have been checked for a possible up-regulation due to the genomic alteration. In 4/5 cell lines established from thyroid adenomas with 19q13.4 rearrangements and 5/5 primary adenomas with that type of rearrangement both the C19MC and miR-371-3 cluster were found to be significantly overexpressed compared to controls lacking that particular chromosome abnormality. In the remaining cell line qRT-PCR revealed overexpression of members of the miR-371-3 cluster only which might be due to a deletion accompanying the chromosomal rearrangement in that case. In depth molecular characterization of the breakpoint in a cell line from one adenoma of this type reveals the existence of large Pol-II mRNA fragments as the most likely source of up-regulation of the C19MC cluster. The up-regulation of the clusters is likely to be causally associated with the pathogenesis of the corresponding tumors. Of note, the expression of miRNAs miR-520c and miR-373 is known to characterize stem cells and in terms of molecular oncology has been implicated in invasive growth of epithelial cells in vitro and in vivo thus allowing to delineate a distinct molecular subtype of thyroid adenomas. Besides thyroid adenomas rearrangements of 19q13.4 are frequently found in other human neoplasias as well, suggesting that activation of both clusters might be a more general phenomenon in human neoplasias

Chromosomal assignment of canine THADA gene to CFA 10q25

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

<p>Abstract</p> <p>Background</p> <p><it>Thyroid adenoma associated (THADA) </it>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 <it>THADA </it>gene expression in normal tissues and in thyroid hyper- and neoplasias, using real-time PCR.</p> <p>Methods</p> <p>For the analysis <it>THADA </it>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, <it>NIS </it>(<it>sodium-iodide symporter</it>) gene expression was measured on 34 of the pathological thyroid samples.</p> <p>Results</p> <p>Results illustrated that <it>THADA </it>expression in normal thyroid tissue was significantly higher (<it>p </it>< 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 (<it>p </it>< 0.001, Wilcoxon test, t approximation), anaplastic carcinomas (ATCs) and all other samples and also between ATCs and all other malignant tumors (<it>p </it>< 0.05, Wilcoxon test, t approximation). Furthermore, in thyroid tumors <it>THADA </it>mRNA expression was found to be inversely correlated with <it>HMGA2 </it>mRNA. <it>HMGA2 </it>expression was recently identified as a marker revealing malignant transformation of thyroid follicular tumors. A correlation between <it>THADA </it>and <it>NIS </it>has also been found in thyroid normal tissue and malignant tumors.</p> <p>Conclusions</p> <p>The results suggest <it>THADA </it>being a marker of dedifferentiation of thyroid tissue.</p

Molecular-cytogenetic and molecular-genetic analysis of thyroid tumors

The focus of the present study was on the molecular-cytogenetic analysis of the main cytogenetic subgroups in follicular lesions of the thyroid. Quantitative molecular-cytogenetic analysis of benign thyroid lesions revealed that much greater importance is attached to the main cytogenetic subgroups, i.e. trisomy 7 and rearrangements of 2p21 or 19q13.4, than by conventional cytogenetic analysis alone. For 2p21-rearrangements it was possible to identify and to further characterize the target gene, THADA, involved in these rearrangements. Further analyses suggest that the truncation of THADA is in correlation with the proliferation of epithelial cells und the genesis of benign thyroid lesions. For 19q13.4 rearrangements the breakpoints were narrowed down to a region of about 150 kb which is in close proximity to two miRNA clusters, C19MC and mir-371-3, which both are activated as a result of the rearrangements. The molecular-cytogenetic analysis of benign as well as malign follicular thyroid neoplasias with respect to the translocation t(2;3)(q13;p25) and the PAX8/PPARγ fusion gene, respectively, revealed that this aberration occurs only in a small fraction of follicular adenomas. Molecular-cytogenetic characterization of the 3p25 breakpoint region reveals that rearrangements of the 3p25 region occur independently of the 2q13 region and the PAX8 gene, respectively. Thus this region is considered as a hot spot region in thyroid neoplasias with follicular origin. In conclusion the results of the present study point out the status of molecular cytogenetics with respect to the analysis of tumor-specific chromosomal aberrations in general and in particular to the relevance of these aberrations to the pathogenesis of benign follicular thyroid lesions

Genomic organization of the fusion gene on the derivative chromosome 1 resulting from a translocation t(1;19)(p35.2;13.4) in cell line S40.2.

<p>Detailed schematic overview illustrating the origin of the fusion transcripts <i>PUM1-FUS-19q-I</i> (Genbank Accession number GQ334687) and PUM1-FUS-19q-II (Genbank Accession number GQ334688) identified in cell line S40.2. The genomic region of PUM1 in 1p35.2 (horizontal gray bar) fuses after exon 10 of <i>PUM1</i> (exons: vertical light gray bars) to the genomic region of <i>C19MC</i> in 19q13.4 (horizontal red bar). The two vertical yellow bars indicate 3′-sequences located after exon 1–10 of <i>PUM1</i> in <i>PUM1-FUS-19q-I</i> and <i>PUM1-FUS-19q-II</i>, respectively, both originating from alternative splicing. The fusion transcripts were detected either by 3′-RACE-PCR (<i>PUM1-FUS-19q-I</i>) or RT-PCR (<i>PUM1-FUS-19q-II</i>) experiments. The quantified miRNAs have been highlighted by their names.</p

Expression analysis of miR-517a by RT-PCR.

<p>PCR reactions were performed and then analyzed in 4% small DNA Agarose. The expected DNA-fragment has a size of 62 bp, Ultra low range Ladder (Fermentas) was used as Marker (M). Lane 1: S40.2, 2: S40.2 without reverse transcriptase (–RT), 3: S121, 4: S121–RT, 5: thyroid (normal), 6: thyroid–RT, 7: placenta, 8: placenta-RT, 9: S270.2, 10: S270.2–RT, 11: S290.1, 12: S290.1–RT, 13: S141.2, 14: S325, 15: S211, 16: S211–RT, 17: fetal RNA, 18: adult testis, 19: fetal RNA-RT, 20: S141.2-RT, 21: adult testis-RT, 22: S325-RT (for details of the cell lines and tumor samples see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009485#pone-0009485-t001" target="_blank">Table 1</a>).</p

Used Primers.

<p>Primer sequences for PCR and cDNA synthesis.</p