Somatic SLC30A1 mutations altering zinc transporter ZnT1 cause aldosterone-producing adenomas and primary aldosteronism.

Primary aldosteronism (PA) is the most common form of endocrine hypertension and is characterized by inappropriately elevated aldosterone production via a renin-independent mechanism. Driver somatic mutations for aldosterone excess have been found in approximately 90% of aldosterone-producing adenomas (APAs). Other causes of lateralized adrenal PA include aldosterone-producing nodules (APNs). Using next-generation sequencing, we identified recurrent in-frame deletions in SLC30A1 in four APAs and one APN (p.L51_A57del, n = 3; p.L49_L55del, n = 2). SLC30A1 encodes the ubiquitous zinc efflux transporter ZnT1 (zinc transporter 1). The identified SLC30A1 variants are situated close to the zinc-binding site (His43 and Asp47) in transmembrane domain II and probably cause abnormal ion transport. Cases of PA with SLC30A1 mutations showed male dominance and demonstrated increased aldosterone and 18-oxocortisol concentrations. Functional studies of the SLC30A151_57del variant in a doxycycline-inducible adrenal cell system revealed pathological Na+ influx. An aberrant Na+ current led to depolarization of the resting membrane potential and, thus, to the opening of voltage-gated calcium (Ca2+) channels. This resulted in an increase in cytosolic Ca2+ activity, which stimulated CYP11B2 mRNA expression and aldosterone production. Collectively, these data implicate zinc transporter alterations as a dominant driver of aldosterone excess in PA.

ß-Catenin-Driven Differentiation Is a Tissue-Specific Epigenetic Vulnerability in Adrenal Cancer.

Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent ß-catenin-activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific ß-catenin-containing complexes, and the epigenome. On chromatin, ß-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, ß-catenin bound histone methyltransferase EZH2. SF1/ß-catenin and EZH2/ß-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/ß-catenin from chromatin and favored EZH2/ß-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities. SIGNIFICANCE: Oncogenic ß-catenin can use tissue-specific partners to regulate cellular differentiation programs that can be reversed by epigenetic therapies, identifying epigenetic control of differentiation as a viable target for ß-catenin-driven cancers.

Development of Human Adrenocortical Adenoma (HAA1) Cell Line from Zona Reticularis.

The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient's macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis.

Targeted Mutational Analysis of Cortisol-Producing Adenomas.

CONTEXT: Somatic gene mutations have been identified in only about half of cortisol-producing adenomas (CPAs). Affected genes include PRKACA, GNAS, PRKAR1A, and CTNNB1. OBJECTIVE: This work aims to expand our understanding of the prevalence of somatic mutations in CPAs from patients with overt Cushing syndrome (OCS) and "subclinical" mild autonomous cortisol excess (MACE), with an immunohistochemistry (IHC)‒guided targeted amplicon sequencing approach using formalin-fixed paraffin-embedded (FFPE) tissue. METHODS: We analyzed FFPE adrenal tissue from 77 patients (n = 12 men, 65 women) with either OCS (n = 32) or MACE (n = 45). Using IHC for 17α-hydroxylase/17,20-lyase (CYP17A1) and 3ß-hydroxysteroid dehydrogenase (HSD3B2), we identified 78 CPAs (32 OCS CPAs and 46 MACE CPAs). Genomic DNA was isolated from the FFPE CPAs and subjected to targeted amplicon sequencing for identification of somatic mutations. RESULTS: Somatic mutations were identified in 71.8% (56/78) of the CPAs. While PRKACA was the most frequently mutated gene in OCS CPAs (14/32, 43.8%), somatic genetic aberrations in CTNNB1 occurred in 56.5% (26/46) of the MACE CPAs. Most GNAS mutations were observed in MACE CPAs (5/7, 71.4%). No mutations were observed in PRKAR1A. In addition to the known mutations, we identified one previously unreported mutation in PRKACA. Two patients with MACE harbored 2 adjacent tumors within the same adrenal gland - one patient had 2 CPAs, and the other patient had a CPA and an aldosterone-producing adenoma (identified by IHC for aldosterone synthase). CONCLUSION: A comprehensive FFPE IHC-guided gene-targeted sequencing approach identified somatic mutations in 71.8% of the CPAs. OCS CPAs demonstrated a distinct mutation profile compared to MACE CPAs.

Transcriptomic Response Dynamics of Human Primary and Immortalized Adrenocortical Cells to Steroidogenic Stimuli.

Adrenal steroid hormone production is a dynamic process stimulated by adrenocorticotropic hormone (ACTH) and angiotensin II (AngII). These ligands initialize a rapid and robust gene expression response required for steroidogenesis. Here, we compare the predominant human immortalized cell line model, H295R cell, with primary cultures of adult adrenocortical cells derived from human kidney donors. We performed temporally resolved RNA-seq on primary cells stimulated with either ACTH or AngII at multiple time points. The magnitude of the expression dynamics elicited by ACTH was greater than AngII in primary cells. This is likely due to the larger population of adrenocortical cells that are responsive to ACTH. The dynamics of stimulus-induced expression in H295R cells are mostly recapitulated in primary cells. However, there are some expression responses in primary cells absent in H295R cells. These data are a resource for the endocrine community and will help researchers determine whether H295R is an appropriate model for the specific aspect of steroidogenesis that they are studying.

Circadian rhythms of 11-oxygenated C19 steroids and ∆5-steroid sulfates in healthy men.

BACKGROUND: Many hormones display distinct circadian rhythms, driven by central regulators, hormonal bioavailability, and half-life. A set of 11-oxygenated C19 steroids (11-oxyandrogens) and pregnenolone sulfate (PregS) are elevated in congenital adrenal hyperplasia and other disorders, but their circadian patterns have not been characterized. PARTICIPANTS AND METHODS: Peripheral blood was collected every 2 h over 24 h from healthy volunteer men (10 young, 18-30 years, and 10 older, 60-80 years). We used mass spectrometry to quantify 15 steroids, including androstenedione (A4), testosterone (T), 11ß-hydroxy- and 11-ketotestosterone (11OHT, 11KT),11ß-hydroxy- and 11-ketoandrostenedione (11OHA4, 11KA4), and 4 ∆5-steroid sulfates. Diurnal models including mesor (rhythm adjusted median), peak, and nadir concentrations, acrophase, and amplitude were computed. RESULTS: 11OHA4 followed a rhythm similar to cortisol: acrophase 8:00 h, nadir 21:00 h and were similar in young and old men. 11KT had similar diurnal patterns, but the peak was lower in older than in young men, as was the case for A4. All four steroid sulfates were higher in young vs older men. PregS and 17-hydroxypregnenolone sulfate (17OHPregS) showed sustained elevations between 8:00 and 18:00 h, and nadirs around midnight, while DHEAS and AdiolS displayed minimal diurnal variations. All 4 11-oxyandrogens correlated tightly with cortisol (r from 0.54 for 11OHT to 0.81 for 11OHA4, P < 0.0001 for all), but very weakly with T, supporting their adrenal origin and ACTH governance. CONCLUSIONS: 11-Oxyandrogens, PregS, and 17OHPregS display distinct circadian and age variations, which should be accounted for when used as clinical biomarkers.

RNA-binding proteins regulate aldosterone homeostasis in human steroidogenic cells.

Angiotensin II (AngII) stimulates adrenocortical cells to produce aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in the precise regulation of AII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding pro-steroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. These data support a model in which coordinated increases in transcription and decay facilitate the major transcriptomic changes required to implement a pro-steroidogenic expression program that actively resolved to prevent aldosterone overproduction.

Intratumoral steroid profiling of adrenal cortisol-producing adenomas by liquid chromatography- mass spectrometry.

Endogenous Cushing syndrome (CS) is an endocrine disorder marked by excess cortisol production rendering patients susceptible to visceral obesity, dyslipidemia, hypertension, osteoporosis and diabetes mellitus. Adrenal CS is characterized by autonomous production of cortisol from cortisol-producing adenomas (CPA) via adrenocorticotropic hormone-independent mechanisms. A limited number of studies have quantified the steroid profiles in sera from patients with CS. To understand the intratumoral steroid biosynthesis, we quantified 19 steroids by mass spectrometry in optimal cutting temperature compound (OCT)-embedded 24 CPA tissue from patients with overt CS (OCS, n = 10) and mild autonomous cortisol excess (MACE, n = 14). Where available, normal CPA-adjacent adrenal tissue (AdjN) was also collected and used for comparison (n = 8). Immunohistochemistry (IHC) for CYP17A1 and HSD3B2, two steroidogenic enzymes required for cortisol synthesis, was performed on OCT sections to confirm the presence of tumor tissue and guided subsequent steroid extraction from the tumor. LC-MS/MS was used to quantify steroids extracted from CPA and AdjN. Our data indicated that CPA demonstrated increased concentrations of cortisol, cortisone, 11-deoxycortisol, corticosterone, progesterone, 17OH-progesterone and 16OH-progesterone as compared to AdjN (p < 0.05). Compared to OCS, MACE patient CPA tissue displayed higher concentrations of corticosterone, 18OH-corticosterone, 21-deoxycortisol, progesterone, and 17OH-progesterone (p < 0.05). These findings also demonstrate that OCT-embedded tissue can be used to define intra-tissue steroid profiles, which will have application for steroid-producing and steroid-responsive tumors.

Aldosterone-Regulating Receptors and Aldosterone-Driver Somatic Mutations.

Background: Somatic gene mutations that facilitate inappropriate intracellular calcium entrance have been identified in most aldosterone-producing adenomas (APAs). Studies suggest that angiotensin II and adrenocorticotropic hormone (ACTH) augment aldosterone production from APAs. Little is known, however, regarding possible variations in response to hormonal stimuli between APAs with different aldosterone-driver mutations. Objective: To analyze the transcript expression of type 1 angiotensin II receptors (AGTR1), ACTH receptors (MC2R), and melanocortin 2 receptor accessory protein (MRAP) in APAs with known aldosterone-driver somatic mutations. Methods: RNA was isolated from APAs with mutations in: KCNJ5 (n = 14), ATP1A1 (n = 14), CACNA1D (n = 14), and ATP2B3 (n = 5), and from normal adjacent adrenal tissue (n = 45). Transcript expression of MC2R, MRAP, AGTR1, aldosterone synthase (CYP11B2), 17α-hydroxylase/17,20-lyase (CYP17A1), and 11ß-hydroxylase (CYP11B1) were quantified using quantitative RT-PCR and normalized to ß-actin. Results: Compared to adjacent normal adrenal tissue, APAs had higher transcript levels of CYP11B2 (2,216.4 [1,112.0, 2,813.5]-fold, p < 0.001), MC2R (2.88 [2.00, 4.52]-fold, p < 0.001), and AGTR1 (1.80 [1.02, 2.80]-fold, p < 0.001]), and lower transcript levels of MRAP, CYP17A1, and CYP11B1 (0.28-0.36, p < 0.001 for all). MC2R and CYP11B2 transcripts were lower in APAs with KCNJ5 vs. other mutations (p < 0.01 for both). MC2R expression correlated positively with that of AGTR1 in APAs harboring KCNJ5 and CACNA1D mutations, and with MRAP expression in APAs harboring ATPase mutations. Conclusions: While MC2R and AGTR1 are expressed in all APAs, differences were observed based on the underlying aldosterone-driver somatic mutations. In tandem, our findings suggest that APAs with ATPase-mutations are more responsive to ACTH than KCNJ5-mutated APAs.

Targeted RNA sequencing of adrenal zones using immunohistochemistry-guided capture of formalin-fixed paraffin-embedded tissue.

Adequate access to fresh or frozen normal adrenal tissue has been a primary limitation to the enhanced characterization of the adrenal zones via RNA sequencing (RNAseq). Herein, we describe the application of targeted RNAseq to formalin-fixed paraffin-embedded (FFPE) normal adrenal gland specimens. Immunohistochemistry (IHC) was used to visualize and guide the capture of the adrenocortical zones and medulla. Following IHC-based tissue capture and isolation of RNA, high-throughput targeted RNAseq highlighted clear transcriptomic differences and identified differentially expressed genes among the adrenal zones. Our data demonstrate the ability to capture FFPE adrenal zone tissue for targeted transcriptomic analyses. Future comparison of normal adrenal zones will improve our understanding of transcriptomic patterns and help identify potential novel pathways controlling zone-specific steroid production.

The Age-Dependent Changes of the Human Adrenal Cortical Zones Are Not Congruent.

BACKGROUND: While previous studies indicate that the zonae reticularis (ZR) and glomerulosa (ZG) diminish with aging, little is known about age-related transformations of the zona fasciculata (ZF). OBJECTIVES: To investigate the morphological and functional changes of the adrenal cortex across adulthood, with emphasis on (i) the understudied ZF and (ii) sexual dimorphisms. METHODS: We used immunohistochemistry to evaluate the expression of aldosterone synthase (CYP11B2), visinin-like protein 1 (VSNL1), 3ß-hydroxysteroid dehydrogenase type II (HSD3B2), 11ß-hydroxylase (CYP11B1), and cytochrome b5 type A (CYB5A) in adrenal glands from 60 adults (30 men), aged 18 to 86. Additionally, we employed mass spectrometry to quantify the morning serum concentrations of cortisol, 11-deoxycortisol (11dF), 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, and androstenedione in 149 pairs of age- and body mass index-matched men and women, age 21 to 95 years. RESULTS: The total cortical area was positively correlated with age (r = 0.34, P = 0.008). Both the total (VSNL1-positive) and functional ZG (CYP11B2-positive) areas declined with aging in men (r = -0.57 and -0.67, P < 0.01), but not in women. The CYB5A-positive area declined with age in both sexes (r = -0.76, P < 0.0001). In contrast, the estimated ZF area correlated positively with age in men (r = 0.59, P = 0.0006) and women (r = 0.49, P = 0.007), while CYP11B1-positive area remained unchanged across ages. Serum cortisol, corticosterone, and 11-deoxycorticosterone levels were stable across ages, while 11dF levels increased slightly with age (r = 0.16, P = 0.007). CONCLUSION: Unlike the ZG and ZR, the ZF and the total adrenal cortex areas enlarge with aging. An abrupt decline of the ZG occurs with age in men only, possibly contributing to sexual dimorphism in cardiovascular risk.

Identification of Somatic Mutations in CLCN2 in Aldosterone-Producing Adenomas.

Somatic mutations driving aldosterone production have been identified in approximately 90% of aldosterone-producing adenomas (APAs) using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided DNA sequencing approach. In the present study, using CYP11B2-guided whole-exome sequencing (WES) and targeted amplicon sequencing, we detected 2 somatic variants in CLCN2 in 2 APAs that were negative for currently known aldosterone-driver mutations. The CLCN2 gene encodes the voltage-gated chloride channel ClC-2. CLCN2 germline variants have previously been shown to cause familial hyperaldosteronism type II. Somatic mutations in CLCN2 were identified in 2 of 115 APAs, resulting in a prevalence of 1.74%. One of the CLCN2 somatic mutations (c.G71A,p.G24D) was identical to a previously described germline variant causing early-onset PA, but was present only as a somatic mutation. The second CLCN2 mutation, which affects the same region of the gene, has not been reported previously (c.64-2_74del). These findings prove that WES of CYP11B2-guided mutation-negative APAs can help determine rarer genetic causes of sporadic PA.

Targeted RNAseq of Formalin-Fixed Paraffin-Embedded Tissue to Differentiate Among Benign and Malignant Adrenal Cortical Tumors.

Lack of routine fresh or frozen tissue is a barrier to widespread transcriptomic analysis of adrenal cortical tumors and an impediment to translational research in endocrinology and endocrine oncology. Our group has previously pioneered the use of targeted amplicon-based next-generation sequencing for archival formalin-fixed paraffin-embedded (FFPE) adrenal tissue specimens to characterize the spectrum of somatic mutations in various forms of primary aldosteronism. Herein, we developed and validated a novel 194-amplicon targeted next-generation RNA sequencing (RNAseq) assay for transcriptomic analysis of adrenal tumors using clinical-grade FFPE specimens. Targeted RNAseq-derived expression values for 27 adrenal cortical tumors, including aldosterone-producing adenomas (APA; n=8), cortisol-producing adenomas (CPA; n=11), and adrenal cortical carcinomas (ACC; n=8), highlighted known differentially-expressed genes (DEGs; i. e., CYP11B2, IGF2, etc.) and tumor type-specific transcriptional modules (i. e., high cell cycle/proliferation transcript expression in ACC, etc.), and a subset of DEGs was validated orthogonally using quantitative reverse transcription PCR (qRT-PCR). Finally, unsupervised hierarchical clustering using a subset of high-confidence DEGs revealed three discrete clusters representing APA, CPA, and ACC tumors with corresponding unique gene expression signatures, suggesting potential clinical utility for a transcriptomic-based approach to tumor classification. Overall, these data support the use of targeted amplicon-based RNAseq for comprehensive transcriptomic profiling of archival FFPE adrenal tumor material and indicate that this approach may facilitate important translational research opportunities for the study of these tumors.

Sex Differences in 11-Oxygenated Androgen Patterns Across Adulthood.

CONTEXT: The gonads are the major source of sex steroids during reproductive ages. The gonadal function declines abruptly in women and gradually in men. The adrenals produce 11-oxygenated androgens (11-oxyandrogens), which start rising during adrenarche. Following menopause, 11-oxyandrogens levels remain similar to reproductive ages. OBJECTIVE: To compare the circulating 11-oxyandrogen concentrations in men and women across adult ages. METHODS: We used mass spectrometry to measure testosterone (T), androstenedione (A4), 11ß-hydroxytestosterone (11OHT), 11-ketotestosterone (11KT), 11ß-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), cortisol, and cortisone in morning sera obtained from adults in outpatient setting. We performed double immunofluorescence of 3ß-hydroxysteroid dehydrogenase type 2 and cytochrome b5 in adrenal tissue from 19 men, age 23-78 years. RESULTS: We included 590 patients (319 men), aged 18 to 97 years, and 84% white. 11KT and 11KA4 were stable across ages in women, but they declined in men (0.21 and 0.06 ng/dL/year, respectively; P < 0.05). 11OHA4 and 11OHT increased modestly with age in women (0.6 and 0.09 ng/dL/year, respectively; P < 0.01), and both remained stable across ages in men. As body mass index (BMI) increased, 11KA4 decreased in women, and 11KT increased in men, both suggesting higher 17ß-hydroxysteroid dehydrogenase activity in obese individuals. A4 and T declined with age and A4 with BMI in both sexes; T declined with BMI in men. Adrenal androgenic enzyme expressions in aging men were similar to those observed in women. CONCLUSIONS: In contrast with traditional androgens, the production of 11OHA4 and 11OHT is sustained with aging in both sexes. The bioactive androgen 11KT declines in aging men but not in women.

Primary aldosteronism diagnostics: KCNJ5 mutations and hybrid steroid synthesis in aldosterone-producing adenomas.

Primary aldosteronism (PA) is characterized by autonomous aldosterone production by renin-independent mechanisms and is most commonly sporadic. While 60-70% of sporadic PA can be attributed to bilateral hyperaldosteronism, the remaining 30-40% is caused by a unilateral aldosterone-producing adenoma (APA). Somatic mutations in or near the selectivity filter the KCNJ5 gene (encoding the potassium channel GIRK4) have been implicated in the pathogenesis of both sporadic and familial PA. Several studies using tumor tissue, peripheral and adrenal vein samples from PA patients have demonstrated that along with aldosterone, the hybrid steroids 18-hydroxycortisol (18OHF) and 18-oxocortisol (18oxoF) are a hallmark of APA harboring KCNJ5 mutations. Herein, we review the recent advances with respect to the molecular mechanisms underlying the pathogenesis of PA and the steroidogenic fingerprints of KCNJ5 mutations. In addition, we present an outlook toward the future of PA subtyping and diagnostic work-up utilizing steroid profiling.

11-Oxygenated androgens in health and disease.

The adrenal gland is a source of sex steroid precursors, and its activity is particularly relevant during fetal development and adrenarche. Following puberty, the synthesis of androgens by the adrenal gland has been considered of little physiologic importance. Dehydroepiandrosterone (DHEA) and its sulfate, DHEAS, are the major adrenal androgen precursors, but they are biologically inactive. The second most abundant unconjugated androgen produced by the human adrenals is 11ß-hydroxyandrostenedione (11OHA4). 11-Ketotestosterone, a downstream metabolite of 11OHA4 (which is mostly produced in peripheral tissues), and its 5α-reduced product, 11-ketodihydrotestosterone, are bioactive androgens, with potencies equivalent to those of testosterone and dihydrotestosterone. These adrenal-derived androgens all share an oxygen atom on carbon 11, so we have collectively termed them 11-oxyandrogens. Over the past decade, these androgens have emerged as major components of several disorders of androgen excess, such as congenital adrenal hyperplasia, premature adrenarche and polycystic ovary syndrome, as well as in androgen-dependent tumours, such as castration-resistant prostate cancer. Moreover, in contrast to the more extensively studied, traditional androgens, circulating concentrations of 11-oxyandrogens do not demonstrate an age-dependent decline. This Review focuses on the rapidly expanding knowledge regarding the implications of 11-oxyandrogens in human physiology and disease.

Molecular and Electrophysiological Analyses of ATP2B4 Gene Variants in Bilateral Adrenal Hyperaldosteronism.

Primary aldosteronism (PA) is the most common cause of secondary hypertension with a high prevalence among patients with resistant hypertension. Despite the recent discovery of somatic variants in aldosterone-producing adenoma (APA)-associated PA, causes for PA due to bilateral aldosterone production (bilateral hyperaldosteronism; BHA) remain unknown. Herein, we identified rare gene variants in ATP2B4, in a cohort of patients with BHA. ATP2B4 belongs to the same family of Ca-ATPases as ATP2B3, which is involved in the pathogenesis of APA. Endogenous ATP2B4 expression was characterized in adrenal tissue, and the gene variants were functionally analyzed for effects on aldosterone synthase (CYP11B2) expression, steroid production in basal and agonist-stimulated conditions, and for changes in biophysical properties of channel properties. Knockdown of ATP2B4 in HAC15 exhibited reduced angiotensin II stimulation in one of four shRNA clones. Stable HAC15 cell lines with doxycycline (dox) - inducible wild-type and variant forms of ATP2B4 - were generated, and dox-induced upregulation of ATP2B4 mRNA and protein was confirmed. However, ATP2B4 variants did not alter basal or agonist-stimulated CYP11B2 expression. Whole-cell recordings in HAC15 cells indicated robust endogenous ATP2B4 conductance in native cells but reduced conductance with overexpressed WT and variant ATP2B4. The previously defined PA-causing ATP2B3 variant served as a positive control and exhibited elevated CYP11B2 mRNA. In conclusion, while this study did not confirm a pathogenic role for ATP2B4 variants in BHA, we describe the sequencing analysis for familial and sporadic BHA and outline a template for the thorough in vitro characterization of gene variants.

Somatic CACNA1H Mutation As a Cause of Aldosterone-Producing Adenoma.

Driver somatic mutations for aldosterone excess have been found in ≈90% of aldosterone-producing adenomas (APAs) using an aldosterone synthase (CYP11B2)-guided sequencing approach. In the present study, we identified a novel somatic CACNA1H mutation (c.T4289C, p.I1430T) in an APA without any currently known aldosterone-driver mutations using CYP11B2 immunohistochemistry-guided whole exome sequencing. The CACNA1H gene encodes a voltage-dependent T-type calcium channel alpha-1H subunit. Germline variants in this gene are known as a cause of familial hyperaldosteronism IV. Targeted next-generation sequencing detected identical CACNA1H variants in 2 additional APAs in a cohort of the University of Michigan, resulting in a prevalence of 4% (3/75) in APAs. We tested the functional effect of the variant on adrenal cell aldosterone production and CYP11B2 mRNA expression using the human adrenocortical HAC15 cell line with a doxycycline-inducible CACNA1HI1430T mutation. Doxycycline treatment increased CYP11B2 mRNA levels as well as aldosterone production, supporting a pathological role of the CACNA1H p.I1430T mutation on the development of primary aldosteronism. In conclusion, somatic CACNA1H mutation is a genetic cause of APAs. Although the prevalence of this mutation is low, this study will provide better understanding of molecular mechanism of inappropriate aldosterone production in APAs.

Chemogenetic activation of adrenocortical Gq signaling causes hyperaldosteronism and disrupts functional zonation.

The mineralocorticoid aldosterone is produced in the adrenal zona glomerulosa (ZG) under the control of the renin-angiotensin II (AngII) system. Primary aldosteronism (PA) results from renin-independent production of aldosterone and is a common cause of hypertension. PA is caused by dysregulated localization of the enzyme aldosterone synthase (Cyp11b2), which is normally restricted to the ZG. Cyp11b2 transcription and aldosterone production are predominantly regulated by AngII activation of the Gq signaling pathway. Here, we report the generation of transgenic mice with Gq-coupled designer receptors exclusively activated by designer drugs (DREADDs) specifically in the adrenal cortex. We show that adrenal-wide ligand activation of Gq DREADD receptors triggered disorganization of adrenal functional zonation, with induction of Cyp11b2 in glucocorticoid-producing zona fasciculata cells. This result was consistent with increased renin-independent aldosterone production and hypertension. All parameters were reversible following termination of DREADD-mediated Gq signaling. These findings demonstrate that Gq signaling is sufficient for adrenocortical aldosterone production and implicate this pathway in the determination of zone-specific steroid production within the adrenal cortex. This transgenic mouse also provides an inducible and reversible model of hyperaldosteronism to investigate PA therapeutics and the mechanisms leading to the damaging effects of aldosterone on the cardiovascular system.

Circulating 11-oxygenated androgens across species.

The androgen precursors, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) are produced in high amounts by the adrenal cortex primarily in humans and a few other primates. The human adrenal also secretes 11-oxygenated androgens (11-oxyandrogens), including 11ß-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), 11ß-hydroxytestosterone (11OHT) and 11-ketotestosterone (11KT), of which 11OHT and 11KT are bioactive androgens. The 11-oxyandrogens, particularly 11KT, have been recognized as biologically important testicular androgens in teleost fishes for decades, but their physiological contribution in humans has only recently been established. Beyond fish and humans, however, the presence of 11-oxyandrogens in other species has not been investigated. This study provides a comprehensive analysis of a set of C19 steroids, including the traditional androgens and 11-oxyandrogens, across 18 animal species. As previously shown, serum DHEA and DHEAS were much higher in primates than all other species. Circulating 11-oxyandrogens, especially 11KT, were observed in notable amounts in male, but not in female trout, consistent with gonadal origin in fish. The circulating concentrations of 11-oxyandrogens ranged from 0.1 to 10 nM in pigs, guinea pigs and in all the primates studied (rhesus macaque, baboon, chimpanzee and human) but not in rats or mice, and 11OHA4 was consistently the most abundant. In contrast to fish, serum 11KT concentrations were similar in male and female primates for each species, despite significantly higher circulating testosterone in males, suggesting that 11KT production in these species is not testis-dependent and primarily originates from adrenal-derived 11-oxyandrogen precursors.