New advances in endocrine hypertension: from genes to biomarkers.

Hypertension (HT) is a major cardiovascular risk factor that affects 10% to 40% of the general population in an age-dependent manner. Detection of secondary forms of HT is particularly important because it allows the targeted management of the underlying disease. Among hypertensive patients, the prevalence of endocrine HT reaches up to 10%. Adrenal diseases are the most frequent cause of endocrine HT and are associated with excess production of mineralocorticoids (mainly primary aldosteronism), glucocorticoids (Cushing syndrome), and catecholamines (pheochromocytoma). In addition, a few rare diseases directly affecting the action of mineralocorticoids and glucocorticoids in the kidney also lead to endocrine HT. Over the past years, genomic and genetic studies have allowed improving our knowledge on the molecular mechanisms of endocrine HT. Those discoveries have opened new opportunities to transfer knowledge to clinical practice for better diagnosis and specific treatment of affected subjects. In this review, we describe the physiology of adrenal hormone biosynthesis and action, the clinical and biochemical characteristics of different forms of endocrine HT, and their underlying genetic defects. We discuss the impact of these discoveries on diagnosis and management of patients, as well as new perspectives related to the use of new biomarkers for improved patient care.

Cluster analysis of angiotensin biomarkers to identify antihypertensive drug treatment in population studies.

BACKGROUND: The recent progress in molecular biology generates an increasing interest in investigating molecular biomarkers as markers of response to treatments. The present work is motivated by a study, where the objective was to explore the potential of the molecular biomarkers of renin-angiotensin-aldosterone system (RAAS) to identify the undertaken antihypertensive treatments in the general population. Population-based studies offer an opportunity to assess the effectiveness of treatments in real-world scenarios. However, lack of quality documentation, especially when electronic health record linkage is unavailable, leads to inaccurate reporting and classification bias. METHOD: We present a machine learning clustering technique to determine the potential of measured RAAS biomarkers for the identification of undertaken treatments in the general population. The biomarkers were simultaneously determined through a novel mass-spectrometry analysis in 800 participants of the Cooperative Health Research In South Tyrol (CHRIS) study with documented antihypertensive treatments. We assessed the agreement, sensitivity and specificity of the resulting clusters against known treatment types. Through the lasso penalized regression, we identified clinical characteristics associated with the biomarkers, accounting for the effects of cluster and treatment classifications. RESULTS: We identified three well-separated clusters: cluster 1 (n = 444) preferentially including individuals not receiving RAAS-targeting drugs; cluster 2 (n = 235) identifying angiotensin type 1 receptor blockers (ARB) users (weighted kappa κw = 74%; sensitivity = 73%; specificity = 83%); and cluster 3 (n = 121) well discriminating angiotensin-converting enzyme inhibitors (ACEi) users (κw = 81%; sensitivity = 55%; specificity = 90%). Individuals in clusters 2 and 3 had higher frequency of diabetes as well as higher fasting glucose and BMI levels. Age, sex and kidney function were strong predictors of the RAAS biomarkers independently of the cluster structure. CONCLUSIONS: Unsupervised clustering of angiotensin-based biomarkers is a viable technique to identify individuals on specific antihypertensive treatments, pointing to a potential application of the biomarkers as useful clinical diagnostic tools even outside of a controlled clinical setting.

Simultaneous sequencing of 37 genes identified causative mutations in the majority of children with renal tubulopathies.

The clinical diagnosis of inherited renal tubulopathies can be challenging as they are rare and characterized by significant phenotypic variability. Advances in sequencing technologies facilitate the establishment of a molecular diagnosis. Therefore, we determined the diagnostic yield of a next generation sequencing panel assessing relevant disease genes in children followed through three national networks with a clinical diagnosis of a renal tubulopathy. DNA was amplified with a kit provided by the European Consortium for High-Throughput Research in Rare Kidney Diseases with nine multiplex PCR reactions. This kit produced 571 amplicons covering 37 genes associated with tubulopathies followed by massive parallel sequencing and bioinformatic interpretation. Identified mutations were confirmed by Sanger sequencing. Overall, 384 index patients and 16 siblings were assessed. Most common clinical diagnoses were 174 patients with Bartter/Gitelman syndrome and 76 with distal renal tubular acidosis. A total of 269 different variants were identified in 27 genes, of which 95 variants were considered likely, 136 definitely pathogenic and 100 had not been described at annotation. These mutations established a genetic diagnosis in 245 of the index patients. Genetic testing changed the clinical diagnosis in 16 cases and provided insights into the phenotypic spectrum of the respective disorders. Our results demonstrate a high diagnostic yield of genetic testing in children with a clinical diagnosis of a renal tubulopathy, consistent with a predominantly genetic etiology in known disease genes. Thus, genetic testing helped establish a definitive diagnosis in almost two-thirds of patients thereby informing prognosis, management and genetic counseling.

MicroRNA-204 Is Necessary for Aldosterone-Stimulated T-Type Calcium Channel Expression in Cardiomyocytes.

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.

WNT/ß-catenin signalling is activated in aldosterone-producing adenomas and controls aldosterone production.

Primary aldosteronism (PA) is the main cause of secondary hypertension, resulting from adrenal aldosterone-producing adenomas (APA) or bilateral hyperplasia. Here, we show that constitutive activation of WNT/ß-catenin signalling is the most frequent molecular alteration found in 70% of APA. We provide evidence that decreased expression of the WNT inhibitor SFRP2 may be contributing to deregulated WNT signalling and APA development in patients. This is supported by the demonstration that mice with genetic ablation of Sfrp2 have increased aldosterone production and ectopic differentiation of zona glomerulosa cells. We further show that ß-catenin plays an essential role in the control of basal and Angiotensin II-induced aldosterone secretion, by activating AT1R, CYP21 and CYP11B2 transcription. This relies on both LEF/TCF-dependent activation of AT1R and CYP21 regulatory regions and indirect activation of CYP21 and CYP11B2 promoters, through increased expression of the nuclear receptors NURR1 and NUR77. Altogether, these data show that aberrant WNT/ß-catenin activation is associated with APA development and suggest that WNT pathway may be a good therapeutic target in PA.

[Genetic alterations in primary aldosteronism]. / Progrès récents dans la génétique de l'hyperaldostéronisme primaire.

Primary aldostéronism (PA) is the most frequent form of arterial hypertension. It is caused in the majority of cases by an aldosterone producing adenoma (APA) of the adrenal cortex or by bilateral adrenal hyperplasia. Recent advances have allowed to identify a certain number of genetic abnormalities involved in the development of APA or responsible for familial forms of PA. These findings have highlighted the central role of calcium signaling in this process. In this review we will discuss the genetic defects associated with PA and discuss the mechanisms whereby they lead to increased aldosterone production and cell proliferation. The possible consequences that this knowledge will have on the diagnosis and management of PA will be addressed.

Dkk3 is a component of the genetic circuitry regulating aldosterone biosynthesis in the adrenal cortex.

Primary aldosteronism (PA, autonomous aldosterone production from the adrenal cortex) causes the most common form of secondary arterial hypertension (HT), which is also the most common curable form of HT. Recent studies have highlighted an important role of mutations in genes encoding potassium channels in the pathogenesis of PA, both in human disease and in animal models. Here, we have exploited the unique features of the hyperaldosteronemic phenotype of Kcnk3 null mice, which is dependent on sexual hormones, to identify genes whose expression is modulated in the adrenal gland according to the dynamic hyperaldosteronemic phenotype of those animals. Genetic inactivation of one of the genes identified by our strategy, dickkopf-3 (Dkk3), whose expression is increased by calcium influx into adrenocortical cells, in the Kcnk3 null background results in the extension of the low-renin, potassium-rich diet insensitive hyperaldosteronemic phenotype to the male sex. Compound Kcnk3/Dkk3 animals display an increased expression of Cyp11b2, the rate-limiting enzyme for aldosterone biosynthesis in the adrenal zona glomerulosa (ZG). Our data show that Dkk3 can act as a modifier gene in a mouse model for altered potassium channel function and suggest its potential involvement in human PA syndromes.

MicroRNAs in aldosterone production and action.

Aldosterone is a cardiovascular hormone with a key role in blood pressure regulation, among other processes, mediated through its targeting of the mineralocorticoid receptor in the renal tubule and selected other tissues. Its secretion from the adrenal gland is a highly controlled process subject to regulatory influence from the renin-angiotensin system and the hypothalamic-pituitary-adrenal axis. MicroRNAs are small endogenous non-coding RNA molecules capable of regulating gene expression post-transcriptionally through stimulation of mRNA degradation or suppression of translation. Several studies have now identified that microRNA levels are changed in cases of aldosterone dysregulation and that microRNAs are capable of regulating the expression of various genes involved in aldosterone production and action. In this article we summarise the major studies concerning this topic. We also discuss the potential role for circulating microRNAs as diagnostic biomarkers for primary aldosteronism, a highly treatable form of secondary hypertension, which would be highly desirable given the current underdiagnosis of this condition.

Macrophages preserve endothelial cell specialization in the adrenal gland to modulate aldosterone secretion and blood pressure.

Macrophages play crucial roles in organ-specific functions and homeostasis. In the adrenal gland, macrophages closely associate with sinusoidal capillaries in the aldosterone-producing zona glomerulosa. We demonstrate that macrophages preserve capillary specialization and modulate aldosterone secretion. Using macrophage-specific deletion of VEGF-A, single-cell transcriptomics, and functional phenotyping, we found that the loss of VEGF-A depletes PLVAP+ fenestrated endothelial cells in the zona glomerulosa, leading to increased basement membrane collagen IV deposition and subendothelial fibrosis. This results in increased aldosterone secretion, called "haptosecretagogue" signaling. Human aldosterone-producing adenomas also show capillary rarefaction and basement membrane thickening. Mice with myeloid cell-specific VEGF-A deletion exhibit elevated serum aldosterone, hypokalemia, and hypertension, mimicking primary aldosteronism. These findings underscore macrophage-to-endothelial cell signaling as essential for endothelial cell specialization, adrenal gland function, and blood pressure regulation, with broader implications for other endocrine organs.

Association of adrenal steroids with metabolomic profiles in patients with primary and endocrine hypertension.

Introduction: Endocrine hypertension (EHT) due to pheochromocytoma/paraganglioma (PPGL), Cushing's syndrome (CS), or primary aldosteronism (PA) is linked to a variety of metabolic alterations and comorbidities. Accordingly, patients with EHT and primary hypertension (PHT) are characterized by distinct metabolic profiles. However, it remains unclear whether the metabolomic differences relate solely to the disease-defining hormonal parameters. Therefore, our objective was to study the association of disease defining hormonal excess and concomitant adrenal steroids with metabolomic alterations in patients with EHT. Methods: Retrospective European multicenter study of 263 patients (mean age 49 years, 50% females; 58 PHT, 69 PPGL, 37 CS, 99 PA) in whom targeted metabolomic and adrenal steroid profiling was available. The association of 13 adrenal steroids with differences in 79 metabolites between PPGL, CS, PA and PHT was examined after correction for age, sex, BMI, and presence of diabetes mellitus. Results: After adjustment for BMI and diabetes mellitus significant association between adrenal steroids and metabolites - 18 in PPGL, 15 in CS, and 23 in PA - were revealed. In PPGL, the majority of metabolite associations were linked to catecholamine excess, whereas in PA, only one metabolite was associated with aldosterone. In contrast, cortisone (16 metabolites), cortisol (6 metabolites), and DHEA (8 metabolites) had the highest number of associated metabolites in PA. In CS, 18-hydroxycortisol significantly influenced 5 metabolites, cortisol affected 4, and cortisone, 11-deoxycortisol, and DHEA each were linked to 3 metabolites. Discussions: Our study indicates cortisol, cortisone, and catecholamine excess are significantly associated with metabolomic variances in EHT versus PHT patients. Notably, catecholamine excess is key to PPGL's metabolomic changes, whereas in PA, other non-defining adrenal steroids mainly account for metabolomic differences. In CS, cortisol, alongside other non-defining adrenal hormones, contributes to these differences, suggesting that metabolic disorders and cardiovascular morbidity in these conditions could also be affected by various adrenal steroids.

Mechanism by Which Inflammation and Oxidative Stress Induce Mineralocorticoid Receptor Gene Expression in Aging Vascular Smooth Muscle Cells.

BACKGROUND: Vascular MR (mineralocorticoid receptor) expression increases with age driving aging-associated vascular stiffness and hypertension. MR has two isoforms (1α and 1ß) with distinct 5'-untranslated and promoter sequences (P1 and P2), but the gene regulatory mechanisms remain unknown. We investigated mechanisms driving MR gene transcriptional regulation in aging human smooth muscle cells (SMC). METHODS: MR was quantified in aortic tissue and primary human aortic SMC (HASMC) comparing adult and aged donors and adult HASMC treated with H2O2, to induce aging. Predicted transcription factor (TF) binding sites in the MR gene were validated using chromatin immunoprecipitations and reporter assays. The impact of TF inhibitors on MR isoforms and fibrosis target gene expression was examined. RESULTS: Expression of both MR mRNA isoforms increased with donor age or H2O2 treatment in HASMCs. HIF1α (hypoxia-inducible factor) and the inflammatory TF NFκB (nuclear factor kappa B) both increased with age in HASMCs and are predicted to bind MR promoters. H2O2 induced HIF1α and NFκB expression and DNA binding of HIF1α to the MR P1 promoter and of NFκB to both MR promoters in HASMCs. HIF1α inhibition decreased MR-1α isoform expression while NFκB inhibition decreased both MR isoforms. HIF1α, NFκB, and MR inhibition decreased the expression of a SMC-MR target gene implicated in vascular fibrosis. In human aortic tissues, expression of HIF1α and NFκB each positively correlated with donor age and MR expression (P<0.0001). CONCLUSIONS: These data implicate the inflammatory TF, NFκB, and oxidative stress-induced TF, HIF1α, in regulating SMC MR transcription in aging HASMCs, which drives aging-related vascular stiffness and cardiovascular disease.

Somatic mutations of CADM1 in aldosterone-producing adenomas and gap junction-dependent regulation of aldosterone production.

Aldosterone-producing adenomas (APAs) are the commonest curable cause of hypertension. Most have gain-of-function somatic mutations of ion channels or transporters. Herein we report the discovery, replication and phenotype of mutations in the neuronal cell adhesion gene CADM1. Independent whole exome sequencing of 40 and 81 APAs found intramembranous p.Val380Asp or p.Gly379Asp variants in two patients whose hypertension and periodic primary aldosteronism were cured by adrenalectomy. Replication identified two more APAs with each variant (total, n = 6). The most upregulated gene (10- to 25-fold) in human adrenocortical H295R cells transduced with the mutations (compared to wildtype) was CYP11B2 (aldosterone synthase), and biological rhythms were the most differentially expressed process. CADM1 knockdown or mutation inhibited gap junction (GJ)-permeable dye transfer. GJ blockade by Gap27 increased CYP11B2 similarly to CADM1 mutation. Human adrenal zona glomerulosa (ZG) expression of GJA1 (the main GJ protein) was patchy, and annular GJs (sequelae of GJ communication) were less prominent in CYP11B2-positive micronodules than adjacent ZG. Somatic mutations of CADM1 cause reversible hypertension and reveal a role for GJ communication in suppressing physiological aldosterone production.

Mineralocorticoid receptor mutations and a severe recessive pseudohypoaldosteronism type 1.

Pseudohypoaldosteronism type 1 (PHA1) is a rare genetic disease of mineralocorticoid resistance characterized by salt wasting and failure to thrive in infancy. Here we describe the first case of a newborn with severe recessive PHA1 caused by two heterozygous mutations in NR3C2, gene coding for the mineralocorticoid receptor (MR). Independent segregation of the mutations occurred in the family, with p.Ser166X being transmitted from the affected father and p.Trp806X from the asymptomatic mother Whereas the truncated MR(166X) protein was degraded, MR(806X) was expressed both at the mRNA and protein level. Functional studies demonstrated that despite its inability to bind aldosterone, MR(806X) had partial ligand-independent transcriptional activity. Partial nuclear localization of MR(806X) in the absence of hormone was identified as a prerequisite to initiate transcription. This exceptional case broadens the spectrum of clinical phenotypes of PHA1 and demonstrates that minimal residual activity of MR is compatible with life. It also suggests that rare hypomorphic NR3C2 alleles may be more common than expected from the prevalence of detected PHA1 cases. This might prove relevant for patient's care in neonatal salt losing disorders and may affect renal salt handling and blood pressure in the general population.

Vascular and hormonal interactions in the adrenal gland.

Primary aldosteronism is the most common form of secondary arterial hypertension, due to excessive aldosterone production from the adrenal gland. Although somatic mutations have been identified in aldosterone producing adenoma, the exact mechanisms leading to increased cell proliferation and nodule formation remain to be established. One hypothesis is that changes in vascular supply to the adrenal cortex, due to phenomena of atherosclerosis or high blood pressure, may influence the morphology of the adrenal cortex, resulting in a compensatory growth and nodule formation in response to local hypoxia. In this review, we will summarize our knowledge on the mechanisms regulating adrenal cortex development and function, describe adrenal vascularization in normal and pathological conditions and address the mechanisms allowing the cross-talk between the hormonal and vascular components to allow the extreme tissue plasticity of the adrenal cortex in response to endogenous and exogenous stimuli. We will then address recent evidence suggesting a role for alterations in the vascular compartment that could eventually be involved in nodule formation and the development of primary aldosteronism.

Colocalization of Wnt/ß-Catenin and ACTH Signaling Pathways and Paracrine Regulation in Aldosterone-producing Adenoma.

CONTEXT: 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. OBJECTIVE: We investigated the role of Wnt/ß-catenin and adrenocorticotropin signaling, as well as elements of paracrine regulation of aldosterone biosynthesis in adrenals with APA and their relationship to intratumoral heterogeneity and mutational status. METHODS: We analyzed the expression of aldosterone-synthase (CYP11B2), CYP17A1, ß-catenin, melanocortin type 2 receptor (MC2R), phosphorlyated cAMP response element-binding protein (pCREB), tryptase, S100, CD34 by multiplex immunofluorescence, and immunohistochemistry-guided reverse transcription-quantitative polymerase chain reaction. Eleven adrenals with APA and 1 with micronodular hyperplasia from patients with PA were analyzed. Main outcome measures included localization of CYP11B2, CYP17A1, ß-catenin, MC2R, pCREB, tryptase, S100, CD34 in APA and aldosterone-producing cell clusters (APCCs). RESULTS: Immunofluorescence revealed abundant mast cells and a dense vascular network in APA, independent of mutational status. Within APA, mast cells were localized in areas expressing CYP11B2 and were rarely colocalized with nerve fibers, suggesting that their degranulation is not controlled by innervation. In these same areas, ß-catenin was activated, suggesting a zona glomerulosa cell identity. In heterogeneous APA with KCNJ5 mutations, MC2R and vascular endothelial growth factor A expression was higher in areas expressing CYP11B2. A similar pattern was observed in APCC, with high expression of CYP11B2, activated ß-catenin, and numerous mast cells. CONCLUSION: Our results suggest that aldosterone-producing structures in adrenals with APA share common molecular characteristics and cellular environment, despite different mutation status, suggesting common developmental mechanisms.

Preanalytical Considerations and Outpatient Versus Inpatient Tests of Plasma Metanephrines to Diagnose Pheochromocytoma.

CONTEXT: Sampling of blood in the supine position for diagnosis of pheochromocytoma and paraganglioma (PPGL) results in lower rates of false positives for plasma normetanephrine than seated sampling. It is unclear how inpatient vs outpatient testing and other preanalytical factors impact false positives. OBJECTIVE: We aimed to identify preanalytical precautions to minimize false-positive results for plasma metanephrines. METHODS: Impacts of different blood sampling conditions on plasma metanephrines were evaluated, including outpatient vs inpatient testing, sampling of blood in semi- vs fully recumbent positions, use of cannulae vs direct venipuncture, and differences in outside temperature. A total of 3147 patients at 10 tertiary referral centers were tested for PPGL, including 278 with and 2869 without tumors. Rates of false-positive results were analyzed. RESULTS: Outpatient rather than inpatient sampling resulted in 44% higher plasma concentrations and a 3.4-fold increase in false-positive results for normetanephrine. Low temperature, a semi-recumbent position, and direct venipuncture also resulted in significantly higher plasma concentrations and rates of false-positive results for plasma normetanephrine than alternative sampling conditions, although with less impact than outpatient sampling. Higher concentrations and rates of false-positive results for plasma normetanephrine with low compared with warm temperatures were only apparent for outpatient sampling. Preanalytical factors were without impact on plasma metanephrines in patients with PPGL. CONCLUSION: Although inpatient blood sampling is largely impractical for screening patients with suspected PPGL, other preanalytical precautions (eg, cannulae, warm testing conditions) may be useful. Inpatient sampling may be reserved for follow-up of patients with difficult to distinguish true- from false-positive results.

Identification of glucocorticoid-related molecular signature by whole blood methylome analysis.

OBJECTIVE: 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. DESIGN: We evaluated whole blood DNA methylome in 94 samples obtained from patients with different glucocorticoid states (Cushing's syndrome, eucortisolism, adrenal insufficiency). We used an independent cohort of 91 samples for validation. METHODS: Leukocyte DNA was obtained from whole blood samples. Methylome was determined using the Illumina methylation chip array (~850 000 CpG sites). Both unsupervised (principal component analysis) and supervised (Limma) methods were used to explore methylome profiles. A Lasso-penalized regression was used to select optimal discriminating features. RESULTS: Whole blood methylation profile was able to discriminate samples by their glucocorticoid status: glucocorticoid excess was associated with DNA hypomethylation, recovering within months after Cushing's syndrome correction. In Cushing's syndrome, an enrichment in hypomethylated CpG sites was observed in the region of FKBP5 gene locus. A methylation predictor of glucocorticoid excess was built on a training cohort and validated on two independent cohorts. Potential CpG sites associated with the risk for specific complications, such as glucocorticoid-related hypertension or osteoporosis, were identified, needing now to be confirmed on independent cohorts. CONCLUSIONS: Whole blood DNA methylome is dynamically impacted by glucocorticoids. This biomarker could contribute to better assessment of glucocorticoid action beyond hormone assays.

Whole blood methylome-derived features to discriminate endocrine hypertension.

BACKGROUND: Arterial hypertension represents a worldwide health burden and a major risk factor for cardiovascular morbidity and mortality. Hypertension can be primary (primary hypertension, PHT), or secondary to endocrine disorders (endocrine hypertension, EHT), such as Cushing's syndrome (CS), primary aldosteronism (PA), and pheochromocytoma/paraganglioma (PPGL). Diagnosis of EHT is currently based on hormone assays. Efficient detection remains challenging, but is crucial to properly orientate patients for diagnostic confirmation and specific treatment. More accurate biomarkers would help in the diagnostic pathway. We hypothesized that each type of endocrine hypertension could be associated with a specific blood DNA methylation signature, which could be used for disease discrimination. To identify such markers, we aimed at exploring the methylome profiles in a cohort of 255 patients with hypertension, either PHT (n = 42) or EHT (n = 213), and at identifying specific discriminating signatures using machine learning approaches. RESULTS: Unsupervised classification of samples showed discrimination of PHT from EHT. CS patients clustered separately from all other patients, whereas PA and PPGL showed an overall overlap. Global methylation was decreased in the CS group compared to PHT. Supervised comparison with PHT identified differentially methylated CpG sites for each type of endocrine hypertension, showing a diffuse genomic location. Among the most differentially methylated genes, FKBP5 was identified in the CS group. Using four different machine learning methods-Lasso (Least Absolute Shrinkage and Selection Operator), Logistic Regression, Random Forest, and Support Vector Machine-predictive models for each type of endocrine hypertension were built on training cohorts (80% of samples for each hypertension type) and estimated on validation cohorts (20% of samples for each hypertension type). Balanced accuracies ranged from 0.55 to 0.74 for predicting EHT, 0.85 to 0.95 for predicting CS, 0.66 to 0.88 for predicting PA, and 0.70 to 0.83 for predicting PPGL. CONCLUSIONS: The blood DNA methylome can discriminate endocrine hypertension, with methylation signatures for each type of endocrine disorder.

Predicting Hypertension Subtypes with Machine Learning Using Targeted Metabolites and Their Ratios.

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.

Identification of risk loci for primary aldosteronism in genome-wide association studies.

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.