Cause Of Primary Aldosteronism Research Articles

Developing the novel diagnostic model and potential drugs by integrating bioinformatics and machine learning for aldosterone-producing adenomas.

Background: Aldosterone-producing adenomas (APA) are a common cause of primary aldosteronism (PA), a clinical syndrome characterized by hypertension and electrolyte disturbances. If untreated, it may lead to serious cardiovascular complications. Therefore, there is an urgent need for potential biomarkers and targeted drugs for the diagnosis and treatment of aldosteronism. Methods: We downloaded two datasets (GSE156931 and GSE60042) from the GEO database and merged them by de-batch effect, then screened the top50 of differential genes using PPI and enriched them, followed by screening the Aldosterone adenoma-related genes (ARGs) in the top50 using three machine learning algorithms. We performed GSEA analysis on the ARGs separately and constructed artificial neural networks based on the ARGs. Finally, the Enrich platform was utilized to identify drugs with potential therapeutic effects on APA by tARGseting the ARGs. Results: We identified 190 differential genes by differential analysis, and then identified the top50 genes by PPI, and the enrichment analysis showed that they were mainly enriched in amino acid metabolic pathways. Then three machine learning algorithms identified five ARGs, namely, SST, RAB3C, PPY, CYP3A4, CDH10, and the ANN constructed on the basis of these five ARGs had better diagnostic effect on APA, in which the AUC of the training set is 1 and the AUC of the validation set is 0.755. And then the Enrich platform identified drugs tARGseting the ARGs with potential therapeutic effects on APA. Conclusion: We identified five ARGs for APA through bioinformatic analysis and constructed Artificial neural network (ANN) based on them with better diagnostic effects, and identified drugs with potential therapeutic effects on APA by tARGseting these ARGs. Our study provides more options for the diagnosis and treatment of APA.

FRI157 Systematic Germline Genetic Analysis In A Cohort Of Adults With Primary Aldosteronism And Hypertension Onset ≤35 yo : Low Yield Of Genetic Pathogenic Variants

Abstract Disclosure: S. Parisien-La Salle: None. D. Nadine: None. Z. El-Haffaf: None. A. Lacroix: None. I. Bourdeau: None. Introduction: Primary aldosteronism (PA) is a common but underdiagnosed cause of hypertension. Although most cases are sporadic, up to 5% of patients have a familial form of PA. Current guidelines recommend germline genetic analysis in patients who present PA < 20 yo or have a family history of PA. As PA is underdiagnosed, we hypothesized that some patients with PA do not undergo germline genetic screening since familial history of PA is unrecognized. Objective: To evaluate the yield of systematic germline genetic analysis for familial PA in patients with PA, hypertension detection ≤ 35 yo and a family history of hypertension. Methods: From 2012-2022, patients diagnosed with PA (positive ARR and confirmatory testing), hypertension onset ≤ 35 yo and a family history of hypertension were offered genetic counselling for germline screening for known familial causes of PA. After informed consent, patients underwent germline analysis for the chimeric gene CYP11B2/CYP11B1 (Ruhr University Bochum, Germany) and a multigene panel including KCNJ5, CACNA1D, CACNA1H and CLCN2 genes (Fulgent, CA). Results: Twenty-five patients consented for genetic analysis. Thirteen patients (52%) were female. In regards to ethnic origin, 52% were French Canadians, 24% African-Americans and the other 24% was split between Asian, European and Middle Eastern descent. The mean age at diagnosis of hypertension was 28.3 yo and 39.7 yo at PA diagnosis. At PA diagnosis, 91.3% (21/23) had previous hypokalemia and 12% (3/25) a cortisol co-secretion (1 mg dexamethasone test> 50 nmol/L). Approximately 13% were on one hypertension drug, 30% on two, 26% on three, 26% on four and 5% were on five medications. Comobordities included: 8% atrial fibrillation, 20% obstructive sleep apnea and 4% coronary heart disease. All patients had a family history of hypertension, 5 patients (20%) had a family history of stroke, and 1 patient (4%) had a family history of PA. Of the patients who had already undergone adrenal vein sampling (n=22), 68.2% (15/22) underwent an adrenalectomy for lateralised aldosterone secretion and 31.8% (7/22) were treated with medical therapy for bilateral disease. None of the 24 patients who underwent testing for glucocorticoid-remediable aldosteronism were found to have the chimeric gene CYP11B2/CYP11B1. Twenty-three patients underwent the multigene panel and no pathogenic variants were identified. However, 4/23 (17,4%) patients carried a variant of unknown significance (VUS) in CACNA1H or CLCN2 genes (CACNA1H c.1315C>T(p.Arg439Cys),CACNA1H c.3868G>A(p.Val1290Ile),CACNA1H c.1939G>A (p.Gly647Ser), CLCN2 c.2402C>T, (p.Thr801Ile)). Conclusion: Systematic germline genetic testing for familial PA in this cohort of patients with PA, hypertension diagnosis ≤ 35 yo and a family history of hypertension did not yield positive results. Although PA is an underdiagnosed disease, familial forms remain very rare. Presentation: Friday, June 16, 2023

Identification of a novel competing endogenous RNA network and candidate drugs associated with ferroptosis in aldosterone-producing adenomas.

Aldosterone-producing adenoma (APA), characterized by unilaterally excessive aldosterone production, is a common cause of primary aldosteronism. Ferroptosis, a recently raised iron-dependent mode of programmed cell death, has been involved in the development and therapy of various diseases. This study obtained datasets of the mRNA and lncRNA expression profiles for APA and adjacent adrenal gland (AAG) from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) and lncRNAs (DE lncRNAs) associated with ferroptosis were identified. Enrichment analyses indicated 89 ferroptosis-related DEGs were primarily enriched in ROS related processes and ferroptosis. Two physical cores, and one combined core were identified in the protein-protein interaction (PPI). DEGs and clinical traits were used in conjunction to screen eight hub genes from two hub modules and 89 DEGs. A competitive endogenous RNA (ceRNA) network was constructed via co-express analysis. Thereafter, molecular docking was used to identify potential targets. Two active compounds, QL-X-138 and MK-1775, bound to AURKA and DUOX1, respectively, with the lowest binding energies. Molecular dynamics simulation verified the stability of the two complexes. In summary, our studies identified eight hub genes and a novel ceRNA regulatory network associated with ferroptosis, wherein QL-X-138 and MK-1775 were considered to be potential drugs for treating APA.

Cardiovascular and metabolic characters of KCNJ5 somatic mutations in primary aldosteronism.

Primary aldosteronism (PA) is the leading cause of curable endocrine hypertension, which is associated with a higher risk of cardiovascular and metabolic insults compared to essential hypertension. Aldosterone-producing adenoma (APA) is a major cause of PA, which can be treated with adrenalectomy. Somatic mutations are the main pathogenesis of aldosterone overproduction in APA, of which KCNJ5 somatic mutations are most common, especially in Asian countries. This article aimed to review the literature on the impacts of KCNJ5 somatic mutations on systemic organ damage. PubMed literature research using keywords combination, including "aldosterone-producing adenoma," "somatic mutations," "KCNJ5," "organ damage," "cardiovascular," "diastolic function," "metabolic syndrome," "autonomous cortisol secretion," etc. APA patients with KCNJ5 somatic mutations are generally younger, female, have higher aldosterone levels, lower potassium levels, larger tumor size, and higher hypertension cure rate after adrenalectomy. This review focuses on the cardiovascular and metabolic aspects of KCNJ5 somatic mutations in APA patients, including left ventricular remodeling and diastolic function, abdominal aortic thickness and calcification, arterial stiffness, metabolic syndrome, abdominal adipose tissue, and correlation with autonomous cortisol secretion. Furthermore, we discuss modalities to differentiate the types of mutations before surgery. KCNJ5 somatic mutations in patients with APA had higher left ventricular mass (LVM), more impaired diastolic function, thicker aortic wall, lower incidence of metabolic syndrome, and possibly a lower incidence of concurrent autonomous cortisol secretion, but better improvement in LVM, diastolic function, arterial stiffness, and aortic wall thickness after adrenalectomy compared to patients without KCNJ5 mutations.

Functional analysis of plasma membrane Ca2+ATPase 3 and its primary aldosteronism-associated mutations.

Primary aldosteronism (PA) is the most common form of secondary hypertension and can lead to higher rates of cardiovascular complications and death than essential hypertension. Aldosterone-producing adenomas (APAs) are the most frequent cause of PA. Most APAs have mutations in plasma membrane ion-transport proteins with 0.6%-9% carrying a mutation in the Plasma Membrane Ca2+ ATPase 3 (PMCA3). We have developed a Xenopus oocyte expression system to measure the function of PMCA3 wildtype (PMCA3WT) and an APA associated deletion mutant (PMCA3L425_V426del.) with minimal endogenous contamination. We measured ATPase activity at 37 °C in plasma membrane preparations from oocytes expressing PMCA3. Ca2+-dependent ATPase was observed in preparations from oocytes expressing PMCA3WT, with a [Ca2+] dependence well described with a rectangular hyperbola (K0.5 = 25.6 ± 5.1 μM) (triplicates from three independent membrane preparations). In contrast, membrane preparations from PMCA3L425_V426del. expressing oocytes lacked Ca2+-dependent ATPase activity, indicating loss of enzymatic activity in the mutant. We used two-electrode voltage clamp electrophysiology to evaluate the presence of PMCA3-variant mediated currents. At resting intracellular [Ca2+], PMCA3WT-injected oocytes had currents indistinguishable from those in uninjected oocytes, at all voltages. When bathed by extracellular 125 mM Na+, PMCA3L425_V426del.-injected oocytes presented inward currents at negative membrane potentials and outward currents at positive ones, consistent with induction of an aberrant channel-like current. The currents were outwardly directed upon substitution of external Na+ with N-methyl D-glucamine+ and were insensitive to changes in extracellular [Ca2+]. These results indicate that PMCA3L425_V426del. causes hyperaldosteronism due to the concomitant loss of active Ca2+ transport and induction of a depolarizing Na+-mediated current. Current experiments are evaluating the ATPase and physiological characteristics of other PA-associated PMCA3 mutations. Funded by NSF-MCB 2003251.

PS-C37-5: NEW THERAPEUTIC PERSPECTIVES FOR BLOOD PRESSURE CONTROL: DEXFADROSTAT PHOSPHATE, A NOVEL ALDOSTERONE SYNTHASE INHIBITOR, IN PATIENTS WITH PRIMARY ALDOSTERONISM

Objective: Aldosterone, the primary mineralocorticoid produced by the adrenal gland, regulates fluid homeostasis by stimulating sodium reabsorption in exchange for potassium in the distal tubules and collecting duct of the kidney. Aldosterone synthase (CYP11B2) is the rate-limiting enzyme for aldosterone biosynthesis. Patients with primary aldosteronism (PA) are characterized by an uncontrolled, excess production of aldosterone and are at higher risk of stroke, atrial fibrillation, and heart failure compared to patients with essential hypertension. Furthermore, PA increases the risk of death even after initiation of medical treatment to control blood pressure. Therefore, PA is the compelling indication to demonstrate the value for an aldosterone synthase inhibitor (ASI) as targeted treatment. Previous attempts to eliminate the excessive spontaneous aldosterone synthesis defining PA have been unsuccessful, particularly due to the unintended inhibition of other steroid hormones. Dexfadrostat phosphate is a novel ASI that acts directly on CYP11B2, targeting the root cause of primary aldosteronism. Design and method: Dexfadrostat phosphate was first investigated in patients with PA diagnosed within 1 year of study entry according to medical guidelines including a suppression test to demonstrate autonomous aldosterone production. Eligibility was verified and approved by a Central Review panel. Following a 2-week placebo-controlled run-in period, enrolled patients were randomized to one of three doses of dexfadrostat taken orally, once daily. After 8 weeks of treatment, patients were switched to placebo and followed for an additional 2 weeks. Blood samples were taken at 2-week intervals and evaluated in a central laboratory for steroid and peptide hormone as well as electrolyte levels. Blood pressure was measured in the office during each clinic visit. Ambulatory systolic blood pressure (aSBP) was monitored over 24-hours at baseline and after 8 weeks of dexfadrostat treatment. Results: All endpoints of the study were met with high significance. Dexfadrostat phosphate treatment was safe and well tolerated. We present the effects on the aldosterone-to-renin ratio (ARR) and subsequent blood pressure reduction, the correction of hypokalemia, and the persistence and reversal of therapeutic effects upon drug withdrawal. Conclusions: The phase 2 study in patients evaluated the ability of dexfadrostat phosphate to deliver both biochemical (ARR, potassium) and subsequent clinical (aSBP) correction of the consequences of uncontrolled aldosterone production. By demonstrating its clinical utility in a rarely diagnosed indication of extreme aldosterone dysregulation, the potential of dexfadrostat phosphate may well expand to address essential hypertension, chronic kidney disease, hypokalemia, volume expansion and organ remodeling.

PS-B04-8: YM750, AN ACAT1 INHIBITOR, REDUCES ALDOSTERONE SECRETION BY DECREASING ADRENAL CYP11B2 EXPRESSION

Primary aldosteronism (PA) is a refractory hypertension that accounts for approximately 5%-10% of hypertensive patients. aldosterone-producing adenomas and idiopathic hyperaldosteronism are known causes of PA. In aldosterone-producing adenomas, depolarization due to somatic mutations of ion channels in adrenocortical cells is considered the cause of aldosterone oversecretion. Recently, there have been reports on the expression of various cholesterol-metabolizing enzymes in aldosterone-producing adenomas, suggesting that intracellular cholesterol metabolism is very important for aldosterone secretion. We present here the possibility that an inhibitor of Acetyl-CoA acetyltransferase (ACAT1), an enzyme that converts cholesterol to cholesteryl esters, may inhibit aldosterone secretion by depolarization in adrenal cells. Potassium chloride and YM750, an ACAT1 inhibitor, were added to human adrenocortical carcinoma, H295R cells. Then, we measured aldosterone synthase gene, CYP11B2 mRNA expression level. As a result, the expression level of CYP11B2 decreased. Both NURR1 and NGFIB mRNA expression levels also decreased. Additionally, aldosterone production was also measured and found to be significantly decreased in the YM750 added group. Next, angiotensin II was added and the inhibitory effect of YM750 was examined. The results showed that CYP11B2 expression was not decreased. This indicates that YM750 decreased the induction of CYP11B2 expression in a potassium chloride-induced stimulus-specific manner. The experimental results suggest that YM750 specifically inhibits CYP11B2 gene expression by cell depolarization and suppresses aldosterone secretion. However, there are few reports of cholesterol metabolism affecting CYP11B2 gene expression. In this study, we found that intracellular cholesterol metabolism may affect CYP11B2 gene expression. We plan to investigate the relationship between ACAT inhibition and CYP11B2 gene expression in detail mechanisms in the future.

PS-B02-14: DISCOVERY OF A NEW DRUG FOR PRIMARY ALDOSTERONISM TARGETING THE CYP11B2 AND KCNJ5 MUTANTS

Primary aldosteronism (PA) is a common cause of secondary hypertension. PA induces damage to cardiovascular and renal blood vessels. Therefore, PA is regarded as a problem because it increases the risk of stroke, cardiomyopathy, kidney disease, etc. Aldosterone-producing adenomas (APA) are one of the major causes of PA, and gene mutations are often identified in tumor cells. KCNJ5 mutation is well known as a gene mutation that causes the loss of ion selectivity in APA. KCNJ5 is an inwardly rectifying potassium channel. KCNJ5 induces sodium ion influx due to mutations in APA. The sodium ion influx induces depolarization, and which overexpresses CYP11B2. CYP11B2 is the rate-determining enzyme for aldosterone synthesis, overexpression of CYP11B2 induces aldosterone overproduction. Examples of therapeutic drugs for PA and APA include calcium channel blockers, aldosterone antagonist and β;-blockers. However, therapeutic drugs for PA and APA targeting cell membrane depolarization have not been developed yet. We aim to find compounds that suppress CYP11B2 expression by membrane depolarization to develop novel therapeutic drugs for PA and APA. By developing a therapeutic drug that targets depolarization, we hope to establish an effective therapeutic method for APA caused by gene mutations such as KCNJ5 mutation. We performed high-throughput screening (HTS) using Tohoku University Compound Library (6080 compounds). First, we generated H295R-CYP11B2 N-Luc cells in which the luciferase gene is inserted downstream of the CYP11B2 locus. The cells H295R-CYP11B2 N-Luc were stimulated with KCl to cause depolarization, after which candidate compounds were added and HTS was performed by Luc assay. As a result, 101 compounds were selected. Furthermore, 25 of 101 compounds were selected by examining the cytotoxicity and dose response. Also, 11 of the 25 compounds suppressed CYP11B2 mRNA expression in KCl stimulated H295R cells. Secondary, we generated KCNJ5 mutant cells that expressed the KCNJ5 mutant (L168R) in a Doxycycline-inducible manner. 3 of 11 compounds that suppressed the expression of CYP11B2 mRNA were selected by examination using KCNJ5 mutant cells. After that, 2 of 3 compounds decreased the aldosterone production. These compounds have a different structure from existing therapeutic drugs such as calcium channel blockers. Since these can be novel therapeutic drugs for PA and APA caused by depolarization such as KCNJ5, we are examing the detailed mechanism of action of the compounds. In addition, we would like to further experiment the effects of the compounds in vivo using hypertensive model mice, Task1 /Task3 double KO mice.

TSPAN12 (Tetraspanin 12) Is a Novel Negative Regulator of Aldosterone Production in Adrenal Physiology and Aldosterone-Producing Adenomas.

Aldosterone-producing adenomas (APAs) are a major cause of primary aldosteronism, a condition of low-renin hypertension, in which aldosterone overproduction is usually driven by a somatic activating mutation in an ion pump or channel. TSPAN12 is differentially expressed in different subgroups of APAs suggesting a role in APA pathophysiology. Our objective was to determine the function of TSPAN12 (tetraspanin 12) in adrenal physiology and pathophysiology. APA specimens, pig adrenals under dietary sodium modulation, and a human adrenocortical cell line HAC15 were used for functional characterization of TSPAN12 in vivo and in vitro. Gene ontology analysis of 21 APA transcriptomes dichotomized according to high versus low TSPAN12 transcript levels highlighted a function for TSPAN12 related to the renin-angiotensin system. TSPAN12 expression levels in a cohort of 30 APAs were inversely correlated with baseline plasma aldosterone concentrations (R=-0.47; P=0.009). In a pig model of renin-angiotensin system activation by dietary salt restriction, TSPAN12 mRNA levels and TSPAN12 immunostaining were markedly increased in the zona glomerulosa layer of the adrenal cortex. In vitro stimulation of human adrenocortical human adrenocortical cells with 10 nM angiotensin II for 6 hours caused a 1.6-fold±0.13 increase in TSPAN12 expression, which was ablated by 10 μM nifedipine (P=0.0097) or 30 μM W-7 (P=0.0022). Gene silencing of TSPAN12 in human adrenocortical cells demonstrated its inverse effect on aldosterone secretion under basal and angiotensin II stimulated conditions. Our findings show that TSPAN12 is a negative regulator of aldosterone production and could contribute to aldosterone overproduction in primary aldosteronism.

OR20-4 Expression of the Neuropeptide Substance P and the NK1 Receptor in Aldosterone-Producing Adenomas

Abstract Background Aldosterone-producing adenoma (APA) is a major cause of primary aldosteronism (PA) which is the most frequent form of secondary hypertension (1-2). The pathophysiology of PA is quite complex and still incompletely understood. Substance P (SP) encoded by the TAC1 gene belongs to a family of bioactive peptides named tachykinins. A recently published study indicated that SP, released by subcapsular nerve fibres, stimulates aldosterone production through activation of the neurokinin type 1 receptor (NK1R) in the human adrenal gland (3). The aim of our work was to investigate the presence of SP fibres and the NK1 receptor in a large series of APA in order to assess the potential role of this peptide in the pathophysiology of PA. Methods APA tissues were studied by molecular and histological approaches. Expression of SP and NK1 receptor was searched for in a series of 51 APA by RT-Q-PCR and immunohistochemistry. Results Quantitative RT-PCR data indicated that adenomas strongly express TAC1 mRNA. Immunohistochemistry showed the presence of SP-positive nerve fibres in APAs tissues. SP was also detected in a subpopulation of adenomatous cells. Adenomas strongly express mRNA encoding the NK1 receptor. The distribution of the NK1 receptor within APAs is similar to that of aldosterone synthase (CYP11B2), suggesting that SP may regulate aldosterone secretion by APAs. Conclusion These results suggest that SP and its NK1 receptor may be involved in the pathophysiology of aldosterone hypersecretion by APAs.

Who should be screened for primary aldosteronism? A comprehensive review of current evidence.

Arterial hypertension is a major risk factor for cardiovascular disease. The prevalence of primary aldosteronism (PA) ranges from 5% to 10% in the general hypertensive population and is regarded as one of the most common causes of secondary hypertension. There are two major causes of PA: bilateral adrenal hyperplasia and aldosterone‐producing adenoma. The diagnosis of PA comprises screening, confirmatory testing, and subtype differentiation. The Endocrine Society Practice Guidelines for the diagnosis and treatment of PA recommends screening of patients at an increased risk of PA. These categories include patients with stage 2 and 3 hypertension, drug‐resistant hypertension, hypertensive with spontaneous or diuretic‐induced hypokalemia, hypertension with adrenal incidentaloma, hypertensive with a family history of early onset hypertension or cerebrovascular accident at a young age, and all hypertensive first‐degree relatives of patients with PA. Recently, several studies have linked PA with obstructive sleep apnea and atrial fibrillation unexplained by structural heart defects and/or other conditions known to cause the arrhythmia, which may be partly responsible for the higher rates of cardiovascular and cerebrovascular accidents in patients with PA. The aim of this review is to discuss which patients should be screened for PA, focusing not only on well‐established guidelines but also on additional groups of patients with a potentially higher prevalence of PA, as has been reported in recent research.

Diagnosis and therapy of primary aldosteronism: new aspects

Primary aldosteronism represents the most frequent cause of endocrine hypertension. It is associated with a higher morbidity and mortality compared to essential hypertension. Early identification of the affected patients is crucial, as the adequate therapy leads to an excellent long-term prognosis, especially after unilateral adrenalectomy. Diagnosis consists of three steps: diagnosis, confirmation test and subtype differentiation. The 2 most frequent causes of primary aldosteronism are aldosterone producing adenoma, which can be cured by surgery, and idiopathic bilateral adrenal hyperplasia, which is treated with mineralocorticoid receptor antagonists.Screening by aldosterone-to-renin ratio is recommended in designed risk populations. As the aldosterone-to-renin ratio displays a limited sensitivity and specificity, confirmatory testing is recommended in most patients with positive screening test. Prediction scores allow to skip confirmatory testing under certain circumstances. Adrenal vein sampling still represents the gold standard in subtype differentiation of primary aldosteronism. Steroid profiling could possibly make dispensable adrenal vein sampling in patients with bilateral hyperplasia. Different studies investigate the potential of functional imaging for differential diagnosis of primary aldosteronism.

Adrenal Vein Sampling in Primary Aldosteronism: A Pictorial Essay for Optimal Left-Side Sampling

Primary aldosteronism (PA) is a curable cause of hypertension. Recent studies have revealed that the actual prevalence of PA is higher than previously recognized. Adrenal vein sampling (AVS) is an essential diagnostic procedure for revealing the cause of PA and determining the treatment plan. The success of AVS is confirmed by comparing cortisol levels between the samples from each adrenal vein and peripheral vein. The failure rate of the procedure is reported to be high in the right adrenal vein, which is directly connected to the inferior vena cava, while that in the left adrenal vein is relatively low; however, this has rarely been reported. In this review, we introduce and analyze cases of failure in left adrenal vein sampling.

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

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. We investigated the role of Wnt/β-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. We analyzed the expression of aldosterone-synthase (CYP11B2), CYP17A1, β-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, β-catenin, MC2R, pCREB, tryptase, S100, CD34 in APA and aldosterone-producing cell clusters (APCCs). 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 β-catenin, and numerous mast cells. 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.

Approaches to Gene Mutation Analysis Using Formalin-Fixed Paraffin-Embedded Adrenal Tumor Tissue From Patients With Primary Aldosteronism.

Aldosterone production is physiologically under the control of circulating potassium and angiotensin II as well as adrenocorticotropic hormone and other secretagogues such as serotonin. The adrenal’s capacity to produce aldosterone relies heavily on the expression of a single enzyme, aldosterone synthase (CYP11B2). This enzyme carries out the final reactions in the synthesis of aldosterone and is expressed almost solely in the adrenal zona glomerulosa. From a disease standpoint, primary aldosteronism (PA) is the most common of all adrenal disorders. PA results from renin-independent adrenal expression of CYP11B2 and production of aldosterone. The major causes of PA are adrenal aldosterone-producing adenomas (APA) and adrenal idiopathic hyperaldosteronism. Our understanding of the genetic causes of APA has significantly improved through comprehensive genetic profiling with next-generation sequencing. Whole-exome sequencing has led to the discovery of mutations in six genes that cause renin-independent aldosterone production and thus PA. To facilitate broad-based prospective and retrospective studies of APA, recent technologic advancements have allowed the determination of tumor mutation status using formalin-fixed paraffin-embedded (FFPE) tissue sections. This approach has the advantages of providing ready access to archival samples and allowing CYP11B2 immunohistochemistry-guided capture of the exact tissue responsible for inappropriate aldosterone synthesis. Herein we review the methods and approaches that facilitate the use of adrenal FFPE material for DNA capture, sequencing, and mutation determination.

X-chromosome variants are associated with aldosterone producing adenomas

Aldosterone-producing adenomas (APAs) are a major cause of primary aldosteronism (PA) and are characterized by constitutively producing aldosterone, which leads to hypertension. Several mutations have been identified in ion channels or ion channel-associated genes that result in APAs. To date, no studies have used a genome-wide association study (GWAS) approach to search for predisposing loci for APAs. Thus, we investigated Scandinavian APA cases (n = 35) and Swedish controls (n = 60) in a GWAS and discovered a susceptibility locus on chromosome Xq13.3 (rs2224095, OR = 7.9, 95% CI = 2.8–22.4, P = 1 × 10–7) in a 4-Mb region that was significantly associated with APA. Direct genotyping of sentinel SNP rs2224095 in a replication cohort of APAs (n = 83) and a control group (n = 740) revealed persistently strong significance (OR = 6.1, 95% CI = 3.5–10.6, p < 0.0005). We sequenced an adjacent gene, MAGEE1, of the sentinel SNP and identified a rare variant in one APA, p.Gly327Glu, which is complementary to other mutations in our primary cohort. Expression quantitative trait loci (eQTL) were investigated on the X-chromosome, and 24 trans-eQTL were identified. Some of the genes identified by trans-eQTL point towards a novel mechanistic explanation for the association of the SNPs with APAs. In conclusion, our study provides further insights into the genetic basis of APAs.

THE 2020 ITALIAN SOCIETY OF HYPERTENSION (SIIA) PRACTICAL GUIDELINES FOR THE MANAGEMENT OF PRIMARY ALDOSTERONISM

Objective: Considering the amount of novel knowledge generated in the last five years, a team of experienced hypertensiologists was assembled to furnish updated clinical practice guidelines for the management of primary aldosteronism (PA). Design and method: They were asked to undertake a systematic review of the literature in international databases by using the PICO strategy, to identify the most relevant studies and to grade their quality. Predefined quality criteria were in compliance with STARD (Standards for Reporting of Diagnostic Accuracy) recommendations. Each section of the guidelines was then jointly prepared by at least two coauthors, who provided, whenever possible, a Class of Recommendation and Level of Evidence, following the American Heart Association (AHA) methodology. The resulting guidelines underwent two rounds of revision, followed by review by an External Committee, two face-to-face meetings with all coauthors to clarify standing queries, a joint presentation on occasion of the 36th SIIA Conference to gather further feedbacks by the all Society members and, on these bases, a further conclusive peer review. Results: As a potentially curable cause of hypertension, PA should always be suspected in hypertensive patients and identified in a cost-effective manner by physicians dealing with these patients. Screening for PA is particularly beneficial when hypertension is severe and/or resistant to treatment, because target treatment and/or surgery allows to control BP, to withdraw or markedly reduce the number and dosage of antihypertensive medications, and to prevent or regress organ damage and associated comorbidities. Subtyping should be performed at third level referral centers that are proficient in performing and interpreting AVS. Adrenalectomy usually cure hyperaldosteronism and hypokalemia when a unilateral cause of PA is discovered; BP can also be normalized or considerably reduced in a substantial proportion of patients. Even when subtyping is unfeasible/failed, identification of PA allows undertaking a target treatment, which will provide better control of high BP. Conclusions: The proposed guidelines, offering substantial updated information, could simplify the diagnosis of PA and assist practicing physicians in the optimization of treatment and follow-up of patients with one of the most common curable causes of arterial hypertension.

GENES MEDIATING CELL GROWTH IN ALDOSTERONE-PRODUCING ADENOMAS

Objective: Aldosterone-producing adenomas (APA) are a major cause of primary aldosteronism. Somatic mutations explain the excess aldosterone production in the majority of patients with APA with mutations in KCNJ5 encoding a potassium channel the most prevalent in most reported populations. Mechanisms driving cell proliferation are largely undefined. Design and method: Quantitative transcriptome analysis using RNA-seq was used to identify differentially expressed genes between macro-APAs (n = 9, diameter &gt;= 30 mm) and micro-APAs (n = 12, diameter &lt;= 10 mm). Validation of RNA-seq data by TaqMan real-time PCR was performed for 14 genes in a broader cohort of APAs (NMD, no mutation detected, n = 28; KCNJ5-mutated, n = 43). Results: Hierarchical cluster analysis of the 500 genes with largest coefficient of variation indicated sample clustering based on genotype (KCNJ5 or NMD) and APA diameter. Differential expression of 155 and 348 genes was found between micro- and macro-APAs with KCNJ5 mutations and NMD, respectively. Among the top 5 overrepresented Gene Ontology terms (biological process), cell death was exclusively enriched in NMD micro- vs NMD macro-APAs. The expression levels of 10 genes were validated with a potential function related to cell growth. Expression of BEX1 (a reported tumour suppressor) was 2.8-fold down regulated in macro-APAs relative to micro-APAs (P &lt; 0.001), and a linear negative correlation of BEX1 expression with APA diameter was observed in NMD APAs (r = -0.501, P = 0.007). Genes involved in beta-catenin signalling, SFRP2, DKK1 and TSPAN12 were 5.9-fold (P &lt; 0.001), 1.8-fold (P = 0.007) and 8.3-fold (P &lt; 0.0001) down regulated, respectively, in macro-APAs compared with micro-APAs. BAI1 and CCL21 were also down regulated in macro-APAs relative to micro-APAs (3.3-fold, P = 0.026; 2.6-fold, P = 0.145, respectively). Additionally, the expression of TMPRSS3 (r = 0.727, P &lt; 0.001), BMP4 (r = 0.389, P = 0.041) and FBXL21 (r = 0.761, P &lt; 0.001) showed a linear positive correlation with adenoma diameter in NMD subgroup. Conclusions: APA display distinct transcriptome profiles according to adenoma diameter which may help identify genes involved in APA tumour cell growth.

BEX1 Is Differentially Expressed in Aldosterone-Producing Adenomas and Protects Human Adrenocortical Cells From Ferroptosis.

[Figure: see text].

Hypertension Editors' Picks: Hyperaldosteronism.

Hypertension Editors' Picks: Hyperaldosteronism.