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SAMPLES (48)

mace:id
Technology # Array version
Species
Organ
Tissue
Physiopathology
Type
Name

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User name	Arndt Benecke
Email	arndt@ihes.fr
Phone / Fax number	+33160926665 / +33160926609
Location	Institut des Hautes Etudes Scientifiques (Integrative Biology) - 35, route de Chartres - 91440 Bures sur Yvette, France

Scientific description

Primary aldosteronism is the major cause of secondary arterial hypertension. Recurrent somatic mutations in KCNJ5, CACNA1D, ATP1A1 and ATP2B3 have been identified in aldosterone producing adenoma (APA). Although the functional link between those mutations and aldosterone production has been clearly established, the mechanisms leading to nodule formation appear to be complex and to partly involve different mechanisms. 
Transcriptomic analysis allowed us to identify the retinoic acid receptor alpha (RAR?) as being a key element in the regulation of adrenal cortex development and function, which may play a role in adrenocortical cell proliferation and nodulation. Consistent with this finding, mice lacking rar? exhibited a structural and functional disorganization of the adrenal cortex, but no major modifications of aldosterone production. This was associated with modifications in the extracellular matrix and vessel architecture. On a molecular level this appeared to be mediated by alterations in Wnt signaling and angiogenesis. 
Our results indicate that retinoic acid receptor signaling contributes to the normal morphology and functional zonation of the adrenal cortex by modulating Wnt signaling as well as VegfA signaling and that any alterations in these pathways could lead to an abnormal cellular proliferation in the adrenal cortex, creating a propitious environment for the emergence of specific driver mutations in APA.

Technical description

Total RNA isolation and labeling. RNA extraction was carried out using a mono-phasic solution of phenol-guanidine isothiocyanate, for maximum recovery, coupled to a cleaning procedure using silica columns for maximum purity. Quality and quantity of the isolated total RNA was determined using an Agilent 2100 Bioanalyzer. 2 to 5 Âµg of mRNA were subjected to RT labeling as instructed by manufacturer (Applied Biosystems, PN 4339628).
Microarray Hybridization and transciptome profiling. Labeled samples were hybridized to Applied Biosystems Human Genome Survey Microarrays V1.0. and V.2.0. Hybridization and chemiluminescence-based detection were done following the protocols supplied by Applied Biosystems (PN 4342142).
Data preprocessing and primary analysis. Acquisition of the chemiluminescence and fluorescence images and primary data analysis were done using Applied Biosystems Expression Array System Software v1.1.1. The primary analysis consists of the following individual operations: (1) Image correction. (2) Global and local background correction. (3) Feature normalization. (4) Spatial normalization. (5) Global normalization. Additionally, we renormalize the resulting data according to the median once more after having removed probes for which the Applied Biosystems Software has set flags equal or greater to 212, indicating compromised or failed measurements, as well as signals from control spots.
Secondary transcriptome analysis. Microarray gene expression data from the primary analysis for each sample was median centered, log 2 transformed, model-adjusted, and quality controlled (Brysbaert et al. 2010). Mean expression profiles for each biological condition were obtained by variance-weighted averaging normalized expression values. Pairwised inter-array normalizations were carried out using the NeONORM method described previously (Noth et al., 2006).  Fold change values were expressed on log2 scale. P-values were determined based on a normal distribution hypothesis of signal intensities using standard methods (Wilhelm et al., 2010). Multiple probes for a single gene, cross-reactivity of a single probe to several genes, as well as the resolution of probe-ID annotations were done as defined previously (Noth & Benecke, 2005). 
Data sets used and nomenclature. Our data set consists of two groups: 11 normal adrenal cortex samples and 48 APA samples, denoted as B_NCC and B_APA, respectively.

Reference

Rami El Zein, Jose Felipe Golib Dzib, Amanda J. Rickard, Fabio Luiz Fernandes-Rosa, Benoit Samson-Couterie, Isabelle Giscos, Angelique Rocha, Marko Pogtlisch, Celso E Gomez-Sanchez, Laurence Amar, Norbert B Ghyselinck, Arndt G. Benecke, Enzo Lalli, Maria-Christina Zennaro, Sheerazed Boulkroun;
Role of retinoic acid signaling in adrenal morphology and functional zonation.