Pheochromocytomas and paragangliomas (PCCs/PGLs) are neural crest-derived tumours with a very strong genetic component. Here we report the first integrated genomic examination of a large collection of PCC/PGL. SNP array analysis reveals distinct copy-number patterns associated with genetic background. Whole-exome sequencing shows a low mutation rate of 0.3 mutations per megabase, with few recurrent somatic mutations in genes not previously associated with PCC/PGL. DNA methylation arrays and miRNA sequencing identify DNA methylation changes and miRNA expression clusters strongly associated with messenger RNA expression profiling. Overexpression of the miRNA cluster 182/96/183 is specific in SDHB-mutated tumours and induces malignant traits, whereas silencing of the imprinted DLK1-MEG3 miRNA cluster appears as a potential driver in a subgroup of sporadic tumours. Altogether, the complete genomic landscape of PCC/PGL is mainly driven by distinct germline and/or somatic mutations in susceptibility genes and reveals different molecular entities, characterized by a set of unique genomic alterations.

Epidemiological reports and studies using rodent models indicate that early exposure to nutrient and/or hormonal challenges can reprogram metabolism at adulthood. Hypothalamic arcuate nucleus (ARC) integrates peripheral and central signals to adequately regulate energy homeostasis. microRNAs (miRNAs) participate in the control of gene expression of large regulatory networks including many signaling pathways involved in epigenetics regulations. Here, we have characterized and compared the miRNA population of ARC of adult male rats continuously exposed to a balanced metabolic environment to the one of adult male rats exposed to an unbalanced high-fat/high-carbohydrate/moderate-protein metabolic environment during the perinatal period and/or at adulthood that consequently displayed hyperinsulinemia and/or hyperleptinemia. We identified more than 400 miRNA species in ARC of adult male rats. By comparing the miRNA content of six biological replicates in each of the four perinatal/adult environments/rat groups, we identified the 10 miRNAs specified by clusters miR-96/182/183, miR-141/200c, and miR-200a/200b/429 as miRNAs of systematic and uncommonly high variation of expression. This uncommon variation of expression may underlie high individual differences in aging disease susceptibilities. By comparing the miRNA content of the adult ARC between the rat groups, we showed that the miRNA population was not affected by the unbalanced adult environment while, in contrast, the expression of 11 miRNAs was repeatedly impacted by the perinatal unbalanced environment. Our data revealed a miRNA response of adult ARC to early metabolic environmental challenge.

Primary aldosteronism (PA) is the most frequent form of secondary arterial hypertension. Mutations in different genes increase aldosterone production in PA, but additional mechanisms may contribute to increased cell proliferation and aldosterone producing adenoma (APA) development. We performed transcriptome analysis in APA and identified retinoic acid receptor alpha (RARα) signaling as a central molecular network involved in nodule formation. To understand how RARα modulates adrenal structure and function, we explored the adrenal phenotype of male and female Rarα knockout mice. Inactivation of Rarα in mice led to significant structural disorganization of the adrenal cortex in both sexes, with increased adrenal cortex size in female mice and increased cell proliferation in males. Abnormalities of vessel architecture and extracellular matrix were due to decreased Vegfa expression and modifications in extracellular matrix components. On the molecular level, Rarα inactivation leads to inhibition of non-canonical Wnt signaling, without affecting the canonical Wnt pathway nor PKA signaling. Our study suggests that Rarα contributes to the maintenance of normal adrenal cortex structure and cell proliferation, by modulating Wnt signaling. Dysregulation of this interaction may contribute to abnormal cell proliferation, creating a propitious environment for the emergence of specific driver mutations in PA.

Catecholamines and adrenocortical steroids are important regulators of blood pressure. Bidirectional relationships between adrenal steroids and catecholamines have been established but whether this is relevant to patients with pheochromocytoma is unclear.

Aldosterone-producing adenomas (APAs) are a common cause of primary aldosteronism (PA). Despite the discovery of somatic mutations in APA and the characterization of multiple factors regulating adrenal differentiation and function, the sequence of events leading to APA formation remains to be determined.

Hypertension is a major global health problem with high prevalence and complex associated health risks. Primary hypertension (PHT) is most common and the reasons behind primary hypertension are largely unknown. Endocrine hypertension (EHT) is another complex form of hypertension with an estimated prevalence varying from 3 to 20% depending on the population studied. It occurs due to underlying conditions associated with hormonal excess mainly related to adrenal tumours and sub-categorised: primary aldosteronism (PA), Cushing's syndrome (CS), pheochromocytoma or functional paraganglioma (PPGL). Endocrine hypertension is often misdiagnosed as primary hypertension, causing delays in treatment for the underlying condition, reduced quality of life, and costly antihypertensive treatment that is often ineffective. This study systematically used targeted metabolomics and high-throughput machine learning methods to predict the key biomarkers in classifying and distinguishing the various subtypes of endocrine and primary hypertension. The trained models successfully classified CS from PHT and EHT from PHT with 92% specificity on the test set. The most prominent targeted metabolites and metabolite ratios for hypertension identification for different disease comparisons were C18:1, C18:2, and Orn/Arg. Sex was identified as an important feature in CS vs. PHT classification.

The Warburg effect describes how cancer cells down-regulate their aerobic respiration and preferentially use glycolysis to generate energy. To evaluate the link between hypoxia and Warburg effect, we studied mitochondrial electron transport, angiogenesis and glycolysis in pheochromocytomas induced by germ-line mutations in VHL, RET, NF1 and SDH genes. SDH and VHL gene mutations have been shown to lead to the activation of hypoxic response, even in normoxic conditions, a process now referred to as pseudohypoxia. We observed a decrease in electron transport protein expression and activity, associated with increased angiogenesis in SDH- and VHL-related, pseudohypoxic tumors, while stimulation of glycolysis was solely observed in VHL tumors. Moreover, microarray analyses revealed that expression of genes involved in these metabolic pathways is an efficient tool for classification of pheochromocytomas in accordance with the predisposition gene mutated. Our data suggest an unexpected association between pseudohypoxia and loss of p53, which leads to a distinct Warburg effect in VHL-related pheochromocytomas.

Primary aldosteronism affects up to 10% of hypertensive patients and is responsible for treatment resistance and increased cardiovascular risk. Here we perform a genome-wide association study in a discovery cohort of 562 cases and 950 controls and identify three main loci on chromosomes 1, 13 and X; associations on chromosome 1 and 13 are replicated in a second cohort and confirmed by a meta-analysis involving 1162 cases and 3296 controls. The association on chromosome 13 is specific to men and stronger in bilateral adrenal hyperplasia than aldosterone producing adenoma. Candidate genes located within the two loci, CASZ1 and RXFP2, are expressed in human and mouse adrenals in different cell clusters. Their overexpression in adrenocortical cells suppresses mineralocorticoid output under basal and stimulated conditions, without affecting cortisol biosynthesis. Our study identifies the first risk loci for primary aldosteronism and highlights new mechanisms for the development of aldosterone excess.

Identification of patients with endocrine forms of hypertension (EHT) (primary hyperaldosteronism [PA], pheochromocytoma/paraganglioma [PPGL], and Cushing syndrome [CS]) provides the basis to implement individualized therapeutic strategies. Targeted metabolomics (TM) have revealed promising results in profiling cardiovascular diseases and endocrine conditions associated with hypertension.

MicroRNAs (miRNAs) modulate gene expression in male germ cells and somatic tissues of mammals on a genome-wide scale. Hundreds of miRNAs are encoded by mammalian genomes, a large fraction of which is expressed in brain. Here we have investigated the complexity and dynamics of miRNA transcriptomes that associate with neuronal network maturation of hypothalamic arcuate nucleus and median eminence (ARC/ME) in rat by analysing more than 300 miRNAs from 3-7 biological replicates at 5 postnatal time-points. The network connecting ARC/ME to other hypothalamic and extra-hypothalamic regions maturates in an environment-dependent manner. We therefore analyzed miRNA transcriptomes of progeny of dams fed either a balanced or unbalanced diet during gestation and lactation. More than 30% of the miRNAs displayed significative changes of expression between stages P8 and P14, and P21 and P28; half of the changes were greater than 3-fold. Among those miRNAs were well-known and dozens of still poorly documented miRNAs. Progeny of dams fed an unbanced diet displayed a severe growth retardation phenotype, lower levels of plasma leptin but almost identical miRNA transcriptomes. Together these data demonstrate that two substantial and robust changes in miRNA transcriptome of ARC/ME occur at a period crucial for neuronal network functional organization.

MicroRNAs (miRNAs) are ∼22-nt molecules exerting control of protein expression in cancer tissues. The current study determined the full spectrum of miRNA dysregulation in freshly isolated human colon or rectal cancer biopsies as well as in controls of healthy adjacent tissue (total of n = 100) using an Illumina sequencing technology. In this work, we aimed to identify miRNAs that may serve as future marker to discern between these two subtypes. DESeq2 analysis revealed 53 significantly dysregulated miRNAs in colon cancer, 67 miRNAs in rectal cancer, and 97 miRNAs in both at a Padj value < 0.05 and ≥ 10 read counts. 65% of miRNAs were upregulated in colon as well as rectal cancer. Highest significant dysregulation (Padj < 0.00001) was detected for hsa-miR-21-5p, -215-5p and -378a in both colon and rectal cancer. Among the group of miRNAs with Padj < 0.05 and more than 2-fold expression differences, hsa-miR-375 was detected in rectal cancer only, and hsa-miR-133a-3p only in colon cancer. Receiver operating characteristic (ROC) analysis confirmed highly distinct sensitivities for hsa-miR-375 to detect rectal cancer (area under the curve (AUC): 0.9), while hsa-miR-133a-3p (AUC: 0.89) had the highest sensitivity for detecting colon cancer. We conclude that hsa-miR-375 and hsa-miR-133a-3p may serve as new markers of rectal or colon cancer and should be further investigated to search for different etiologies of colorectal cancer.

Fibromuscular dysplasia (FMD) and spontaneous coronary artery dissection (SCAD) are related, non-atherosclerotic arterial diseases mainly affecting middle-aged women. Little is known about their physiopathological mechanisms. We aimed to identify rare genetic causes to elucidate molecular mechanisms implicated in FMD and SCAD.

Activation of the mineralocorticoid receptor (MR) in the heart is considered to be a cardiovascular risk factor. MR activation leads to heart hypertrophy and arrhythmia. In ventricular cardiomyocytes, aldosterone induces a profound remodeling of ion channel expression, in particular, an increase in the expression and activity of T-type voltage-gated calcium channels (T-channels). The molecular mechanisms immediately downstream from MR activation, which lead to the increased expression of T-channels and, consecutively, to an acceleration of spontaneous cell contractions in vitro, remain poorly investigated. Here, we investigated the putative role of a specific microRNA in linking MR activation to the regulation of T-channel expression and cardiomyocyte beating frequency. A screening assay identified microRNA 204 (miR-204) as one of the major upregulated microRNAs after aldosterone stimulation of isolated neonatal rat cardiomyocytes. Aldosterone significantly increased the level of miR-204, an effect blocked by the MR antagonist spironolactone. When miR-204 was overexpressed in isolated cardiomyocytes, their spontaneous beating frequency was significantly increased after 24 h, like upon aldosterone stimulation, and messenger RNAs coding T-channels (CaV3.1 and CaV3.2) were increased. Concomitantly, T-type calcium currents were significantly increased upon miR-204 overexpression. Specifically repressing the expression of miR-204 abolished the aldosterone-induced increase of CaV3.1 and CaV3.2 mRNAs, as well as T-type calcium currents. Finally, aldosterone and miR-204 overexpression were found to reduce REST-NRSF, a known transcriptional repressor of CaV3.2 T-type calcium channels. Our study thus strongly suggests that miR-204 expression stimulated by aldosterone promotes the expression of T-channels in isolated rat ventricular cardiomyocytes, and therefore, increases the frequency of the cell spontaneous contractions, presumably through the inhibition of REST-NRSF protein.

Cushing's syndrome represents a state of excessive glucocorticoids related to glucocorticoid treatments or to endogenous hypercortisolism. Cushing's syndrome is associated with high morbidity, with significant inter-individual variability. Likewise, adrenal insufficiency is a life-threatening condition of cortisol deprivation. Currently, hormone assays contribute to identify Cushing's syndrome or adrenal insufficiency. However, no biomarker directly quantifies the biological glucocorticoid action. The aim of this study was to identify such markers.

Enduring behavioral changes upon stress exposure involve changes in gene expression sustained by epigenetic modifications in brain circuits, including the mesocorticolimbic pathway. Brahma (BRM) and Brahma Related Gene 1 (BRG1) are ATPase subunits of the SWI/SNF complexes involved in chromatin remodeling, a process essential to enduring plastic changes in gene expression. Here, we show that in mice, social defeat induces changes in BRG1 nuclear distribution. The inactivation of the Brg1/Smarca4 gene within dopamine-innervated regions or the constitutive inactivation of the Brm/Smarca2 gene leads to resilience to repeated social defeat and decreases the behavioral responses to cocaine without impacting midbrain dopamine neurons activity. Within striatal medium spiny neurons, Brg1 gene inactivation reduces the expression of stress- and cocaine-induced immediate early genes, increases levels of heterochromatin and at a global scale decreases chromatin accessibility. Altogether these data demonstrate the pivotal function of SWI/SNF complexes in behavioral and transcriptional adaptations to salient environmental challenges.

Objective structured clinical examinations (OSCE) evaluate clinical reasoning, communication skills, and interpersonal behavior during medical education. In France, clinical training has long relied on bedside clinical practice in academic hospitals. The need for a simulated teaching environment has recently emerged, due to the increasing number of students admitted to medical schools, and the necessity of objectively evaluating practical skills. This study aimed at investigating the relationships between OSCE grades and current evaluation modalities.

Medication adherence plays a critical role in controlling the evolution of chronic disease, as low medication adherence may lead to worse health outcomes, higher mortality, and morbidity. Assessment of their patients' medication adherence by clinicians is essential for avoiding inappropriate therapeutic intensification, associated health care expenditures, and the inappropriate inclusion of patients in time- and resource-consuming educational interventions. In both research and clinical practices the most extensively used measures of medication adherence are patient-reported outcome measures (PROMs), because of their ability to capture subjective dimensions of nonadherence. Machine learning (ML), a subfield of artificial intelligence, uses computer algorithms that automatically improve through experience. In this context, ML tools could efficiently model the complexity of and interactions between multiple patient behaviors that lead to medication adherence.

Primary aldosteronism (PA) is the most common form of secondary hypertension. Mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D are found in aldosterone producing adenoma (APA) and familial hyperaldosteronism (FH). A recurrent mutation in CACNA1H (coding for Cav3.2) was identified in a familial form of early onset PA. Here we performed whole exome sequencing (WES) in patients with different types of PA to identify new susceptibility genes. Four different heterozygous germline CACNA1H variants were identified. A de novo Cav3.2 p.Met1549Ile variant was found in early onset PA and multiplex developmental disorder. Cav3.2 p.Ser196Leu and p.Pro2083Leu were found in two patients with FH, and p.Val1951Glu was identified in one patient with APA. Electrophysiological analysis of mutant Cav3.2 channels revealed significant changes in the Ca2+ current properties for all mutants, suggesting a gain of function phenotype. Transfections of mutant Cav3.2 in H295R-S2 cells led to increased aldosterone production and/or expression of genes coding for steroidogenic enzymes after K+ stimulation. Identification of CACNA1H mutations associated with early onset PA, FH, and APA suggests that CACNA1H might be a susceptibility gene predisposing to PA with different phenotypic presentations, opening new perspectives for genetic diagnosis and management of patients with PA.

Paragangliomas are neuroendocrine tumors frequently associated with mutations in RET, NF1, VHL, and succinate dehydrogenase (SDHx) genes. Methylome analysis of a large paraganglioma cohort identified three stable clusters, associated with distinct clinical features and mutational status. SDHx-related tumors displayed a hypermethylator phenotype, associated with downregulation of key genes involved in neuroendocrine differentiation. Succinate accumulation in SDH-deficient mouse chromaffin cells led to DNA hypermethylation by inhibition of 2-OG-dependent histone and DNA demethylases and established a migratory phenotype reversed by decitabine treatment. Epigenetic silencing was particularly severe in SDHB-mutated tumors, potentially explaining their malignancy. Finally, inactivating FH mutations were identified in the only hypermethylated tumor without SDHx mutations. These findings emphasize the interplay between the Krebs cycle, epigenomic changes, and cancer.

Rationale: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through in vitro models, and define specific therapeutic options according to tumor genomic features. Methods: We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized in vitro. Results: A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients' liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, P=4.67·10-18), and was found associated in vitro with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated in vitro a TSC2 repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. Conclusions: Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients' management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors.