Transcriptome, Methylome and Genomic Variations Analysis of Ectopic Thyroid Glands

Congenital hypothyroidism from thyroid dysgenesis (CHTD) is predominantly a sporadic disease characterized by defects in the differentiation, migration or growth of thyroid tissue. Of these defects, incomplete migration resulting in ectopic thyroid tissue is the most common (up to 80%). Germinal mutations in the thyroid-related transcription factors NKX2.1, FOXE1, PAX-8, and NKX2.5 have been identified in only 3% of patients with sporadic CHTD. Moreover, a survey of monozygotic twins yielded a discordance rate of 92%, suggesting that somatic events, genetic or epigenetic, probably play an important role in the etiology of CHTD.Journal ArticleResearch Support, Non-U.S. Gov'tValidation StudiesSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Small amplified RNA-SAGE: an alternative approach to study transcriptome from limiting amount of mRNA

Serial analysis of gene expression (SAGE) is a widely used and powerful technique to characterize and compare transcriptomes. Although several modifications have been proposed to the initial protocol with the aim of reducing the amount of starting material, unless additional PCR steps are added, the technique is still limited by the need for at least 1 µg of total RNA. As extra PCR amplification might introduce representation biases, current SAGE protocols are not fully suitable for the study of small, microdissected tissue samples. We propose here an alternative method involving the linear amplification of small mRNA fragments containing the SAGE tags. The procedure allows preparation of libraries of over 100 000 tags from as few as 2500 cells. A satisfactory correlation was observed between a microSAGE library made from 5 µg of total thyroid RNA, and a library prepared from 50 ng of the same RNA preparation according to the present protocol

Regulated Activating Thr172 Phosphorylation of Cyclin-Dependent Kinase 4(CDK4): Its Relationship with Cyclins and CDK “Inhibitors”

Cyclin-dependent kinase 4 (CDK4) is a master integrator of mitogenic and antimitogenic extracellular signals. It is also crucial for many oncogenic transformation processes. Various molecular features of CDK4 activation remain poorly known or debated, including the regulation of its association with D-type cyclins, its activating Thr172 phosphorylation, and the roles of Cip/Kip CDK “inhibitors” in these processes. Thr172 phosphorylation of CDK4 was reinvestigated using two-dimensional gel electrophoresis in various experimental systems, including human fibroblasts, canine thyroid epithelial cells stimulated by thyrotropin, and transfected mammalian and insect cells. Thr172 phosphorylation of CDK4 depended on prior D-type cyclin binding, but Thr172 phosphorylation was also found in p16-bound CDK4. Opposite effects of p27 on cyclin D3-CDK4 activity observed in different systems depended on its stoichiometry in this complex. Thr172-phosphorylated CDK4 was enriched in complexes containing p21 or p27, even at inhibitory levels of p27 that precluded CDK4 activity. Deletion of the p27 nuclear localization signal sequence relocalized cyclin D3-CDK4 in the cytoplasm but did not affect CDK4 phosphorylation. Within cyclin D3 complexes, T-loop phosphorylation of CDK4, but not of CDK6, was directly regulated, identifying it as a determining target for cell cycle control by extracellular factors. Collectively, these unexpected observations indicate that CDK4-activating kinase(s) should be reconsidered

Epigenetic basis for monocyte dysfunction in patients with severe alcoholic hepatitis

International audienceBackground & aims: Severe forms of alcohol-related liver disease are associated with increased susceptibility to infections which are associated with poor prognosis. The cellular and molecular mechanisms responsible for this altered host defense are incompletely understood.Methods: We performed whole blood phenotypic analysis and ex vivo stimulation with various pathogen-associated molecular patterns (PAMPs). We included 34 patients with alcohol-related cirrhosis (18 of whom had biopsy-proven severe alcoholic hepatitis [sAH]), 12 healthy controls and 11 patients with chronic alcohol consumption without significant liver disease. We also evaluated the transcriptomic (RNA-seq) and chromatin accessibility (ATAC-seq) profiles of CD14+ monocytes from a subset of patients.Results: Circulating monocytes and conventional dendritic cells (DCs) from patients with sAH displayed complex alterations characterized by increased expression of both activating and inhibitory surface markers and an impaired pro-inflammatory response upon stimulation with PAMPs representative of gram-negative bacteria (lipopolysaccharide, Pam3CSK4) or fungal pathogens (Zymosan). Their decreased ability to produce more than 1 cytokine (polyfunctionality) upon PAMP stimulation correlated with the risk of developing infection at 28 days or mortality at 90 days. The presence of acute-on-chronic liver failure in patients with sAH did not significantly modify the immune profile of monocytes and DCs. Moreover, CD14+ monocytes of patients with sAH displayed altered transcriptional and epigenomic profiles characterized by downregulation of key innate immune and metabolic pathways and upregulation of important immunomodulatory factors.Conclusions: In patients with sAH, the altered transcriptional program and functional properties of monocytes that contribute to patients' susceptibility to infection have strong epigenetic determinants.Lay summary: Patients with severe alcoholic hepatitis are at increased risk of infections, which contribute to the poor prognosis associated with the disease. Herein, we show that epigenetic determinants underly the immune cell dysfunction and inappropriate responses to pathogens that are associated with severe alcoholic hepatitis

Genotypic and gene expression studies in congenital melanocytic nevi: insight into initial steps of melanotumorigenesis

Large congenital melanocytic nevi (CMNs) are said to have a higher propensity to malignant transformation compared with acquired nevi. Thus, they represent a good model for studying initial steps of melanotumorigenesis. We have performed genotypic (karyotype, fluorescence in situ hybridization, and mutational analyses) and differential expression studies on a large cohort of medium (n=3) and large (n=24) CMN. Chromosomal abnormalities were rare and single, a feature probably reflecting the benignity of these lesions. Mutational screening showed a high frequency of NRAS mutations in our series (19/27 cases, 70%), whereas BRAF mutations were less common (4/27 cases, 15%). Differential did not show significant alterations of cellular processes such as cell proliferation, cell migration/invasion, angiogenesis, apoptosis, and immune/inflammatory responses. However, significant downregulation of genes involved in pigmentation and upregulation of genes playing a role in DNA protection were observed. Lastly, our microarrays displayed upregulation of genes mediating chemoresistance in cancer. As alteration of pigmentation mechanisms can trigger oxidative damage, increased expression of genes involved in maintenance of DNA integrity might reflect the ability of nevocytic cells to self-protect against cellular stress. Furthermore, the observed alterations linked to chemoresistance might partially account for the well-known inefficacy of chemotherapy in malignant melanoma

Principles Governing A-to-I RNA Editing in the Breast Cancer Transcriptome

Little is known about how RNA editing operates in cancer. Transcriptome analysis of 68 normal and cancerous breast tissues revealed that the editing enzyme ADAR acts uniformly, on the same loci, across tissues. In controlled ADAR expression experiments, the editing frequency increased at all loci with ADAR expression levels according to the logistic model. Loci-specific “editabilities,” i.e., propensities to be edited by ADAR, were quantifiable by fitting the logistic function to dose-response data. The editing frequency was increased in tumor cells in comparison to normal controls. Type I interferon response and ADAR DNA copy number together explained 53% of ADAR expression variance in breast cancers. ADAR silencing using small hairpin RNA lentivirus transduction in breast cancer cell lines led to less cell proliferation and more apoptosis. A-to-I editing is a pervasive, yet reproducible, source of variation that is globally controlled by 1q amplification and inflammation, both of which are highly prevalent among human cancers

Hierarchical clustering of the microarray data from the 6 cell lines and from different malignant thyroid tumors (PTC, FTC and ATC).

<p>Hierarchical clustering was generated using R 2.14.1. The method used to compute the distance was “Ward”. All the cell lines clustered together, with the ATC showing that the cell lines are closest to the dedifferentiated tumors than the tumors from which they derived. The data from the six cell lines were the mean of triplicate experiments. Differentiated tumors refer to papillary (PTC) and follicular (FTC) thyroid carcinomas.</p

Heatmap based on the results of SAM one class analysis of the microarray data from 6 thyroid cancer cell lines and compared to tumor tissues.

<p>18 of the 19 modulated miRNA in cell lines are modulated in the same way in ATC, but are not modulated or in an opposite way in differentiated tumors (PTC and FTC). The heatmap was generated using R 2.14.1. with the library “gplots”. ATC.M (n = 11), PTC.M (n = 8) and FTC.M (n = 8) refer to the mean of the miRNA data from the different tumors analyzed. * miRNA described as regulated in ATC by Braun et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111581#pone.0111581-Braun1" target="_blank">[23]</a>. # miRNA described as regulated in ATC by Visone et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111581#pone.0111581-Visone1" target="_blank">[24]</a>.</p

Hierarchical clustering on the miRNA expression data from primary cultures treated with TSH or with EGF/serum and from tumor tissues.

<p>Hierarchical clusterings were generated using R 2.8.1. The method used to compute the distance was “Ward”. A. Hierarchical clustering on the data from the primary cultured thyrocytes stimulated with 0.3 mU/ml of TSH for 1.5, 3, 16, 24, 48 and 72 hours and from 7 autonomous hyperfunctioning adenomas (AA). A time evolution in miRNA expression according to TSH treatment of primary cultured thyrocytes is observed suggesting fine changes in miRNA level. The AA cluster apart from the TSH treated primary cultures indicating a clear separation between those two groups of samples. Each time point is the mean of triplicates. B. Hierarchical clustering on the data from primary cultured thyrocytes stimulated with 25 ng/ml EGF and 10% serum for 1.5, 3, 16, 24, 48 and 72 hours and from 8 papillary thyroid carcinomas. In this case, no temporal evolution could be observed by hierarchical clustering. Each time point is the mean of triplicates. Tumors and treated primary cultures clustered apart, suggesting that primary cultures evolved differently according to the treatment but that there was no convergence to AA or PTC for the long-term treatment.</p