Publications by authors named "William E Rainey"

180 Publications

Circadian Rhythms of 11-oxygenated C19 Steroids and ∆5-Steroid Sulfates in Healthy Men.

Eur J Endocrinol 2021 Jul 1. Epub 2021 Jul 1.

R Auchus, Pharmacology & Internal Medicine, Division of Metabolism, Endocrinology & Diabetes, University of Michigan, Ann Arbor, United States.

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), 11β-hydroxy- and 11-ketotestosterone (11OHT, 11KT),11β-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, 8AM, nadir, 9PM 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 4 steroid sulfates were higher in young vs. older men. PregS and 17-hydroxypregnenolone sulfate (17OHPregS) showed sustained elevations between 8AM-6PM, 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.
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http://dx.doi.org/10.1530/EJE-21-0348DOI Listing
July 2021

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

Front Endocrinol (Lausanne) 2021 29;12:683588. Epub 2021 Jun 29.

Department of Pathology, University of Michigan, Ann Arbor, MI, United States.

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.
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http://dx.doi.org/10.3389/fendo.2021.683588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8276099PMC
June 2021

Concomitant Pheochromocytoma and Primary Aldosteronism: A Case Series and Literature Review.

J Endocr Soc 2021 Aug 16;5(8):bvab107. Epub 2021 Jun 16.

Division of Endocrinology, Diabetes, Metabolism, and Nutrition, Mayo Clinic, Rochester, MN, USA.

Context: The detection and management of concomitant pheochromocytoma (PHEO) and primary aldosteronism (PA) is not well understood.

Objective: To investigate varying presentations and outcomes of cases with coexisting PHEO and PA to provide an approach to its diagnosis and management.

Methods: We conducted a retrospective case series of adult patients with concomitant PHEO and PA at Mayo Clinic from 2000-2020 and an additional review of cases before 2000 and from the medical literature. Clinical, biochemical, radiologic, and histologic parameters were measured.

Results: Fifteen patients (53% men, median age 53 years) were diagnosed with concomitant PHEO and PA. The majority presented with hypertension (13, 87%) and hypokalemia (13, 87%), and 6 (40%) presented with symptoms suggestive of catecholamine excess. All patients who underwent preoperative workup for catecholamine excess (14, 93%) were found to have biochemical levels above the upper limits of normal. Adrenal vein sampling (AVS) was performed in 9 patients (60%), where 5 (56%) were diagnosed with bilateral PA, and 4 (44%) with unilateral PA. Patients underwent either unilateral (12, 80%) or bilateral (3, 20%) adrenalectomy. Biochemical improvement or resolution of catecholamine excess was confirmed in all cases with documented measurements. Recurrence of PHEO was not observed. Six patients (40%) displayed persistent PA postoperatively.

Conclusion: Concomitant PHEO and PA is a rare but likely underreported condition. Hypertension with or without hypokalemia should prompt evaluation for PA, while any indeterminate adrenal mass should be assessed for PHEO. Coexisting disease warrants consideration of AVS to determine the laterality of PA to ensure appropriate management.
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http://dx.doi.org/10.1210/jendso/bvab107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8271195PMC
August 2021

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

J Steroid Biochem Mol Biol 2021 Jun 2;212:105924. Epub 2021 Jun 2.

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, 48109, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jsbmb.2021.105924DOI Listing
June 2021

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

RNA 2021 Jun 1. Epub 2021 Jun 1.

University of Colorado Denver School of Medicine;

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.
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http://dx.doi.org/10.1261/rna.078727.121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284322PMC
June 2021

Single-Center Prospective Cohort Study on the Histopathology, Genotype, and Postsurgical Outcomes of Patients With Primary Aldosteronism.

Hypertension 2021 May 24:HYPERTENSIONAHA12117348. Epub 2021 May 24.

Medizinische Klinik und Poliklinik IV, Klinikum der Universitaät Muünchen, Ludwig-Maximilians-Universität München, Germany. (L.S.M., L.H., I.K., M.B., M.R., T.A.W.).

Unilateral forms of primary aldosteronism are usually surgically treated to remove the source of aldosterone excess. After adrenalectomy, aldosteronism persists in some patients indicating abnormal aldosterone production from the unresected gland. Our objective was to investigate histopathology, genotype, and postsurgical outcomes in a 3-year prospective cohort of surgically treated patients for primary aldosteronism (from 2016 to 2018). The cohort comprised 60 consecutively operated patients categorized with classical or nonclassical histopathologic findings of unilateral primary aldosteronism. In the classical group were 45 solitary aldosterone-producing adenomas or dominant aldosterone-producing nodules; in the nonclassical group were 15 cases of multiple aldosterone-producing micronodules or nodules (12 cases) or aldosterone-producing diffuse hyperplasia (3 cases). The classical group displayed higher baseline plasma aldosterone concentrations (262 versus 155 pg/mL, =0.008) and an increased aldosterone-to-renin ratio (81 versus 42, =0.002). A high proportion of the classical group achieved complete biochemical success (97.6% versus 66.7% in the nonclassical group, =0.002). The nonclassical versus classical group displayed an increased ratio of absolute aldosterone concentration in the contralateral adrenal vein to peripheral vein at adrenal venous sampling (3.8 versus 2.0, =0.004). Variants in aldosterone-driver genes were identified in 85% of 41 aldosterone-producing adenomas and were excluded in the remaining 15% by CYP11B2 guided next-generation sequencing. There were no differences in clinical or biochemical outcomes in patients with a solitary aldosterone-producing adenoma categorized by mutation status. In conclusion, adrenals with a nonclassical histopathology of unilateral primary aldosteronism are associated with a higher incidence of postsurgical disease persistence and increased aldosterone production from the unresected adrenal.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.121.17348DOI Listing
May 2021

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

Mol Cell Endocrinol 2021 06 26;530:111296. Epub 2021 Apr 26.

Michigan Center for Translational Pathology, Ann Arbor, MI, USA; Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA; Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.mce.2021.111296DOI Listing
June 2021

GENETICS IN ENDOCRINOLOGY: Impact of race and sex on genetic causes of aldosterone-producing adenomas.

Eur J Endocrinol 2021 May 21;185(1):R1-R11. Epub 2021 May 21.

Departments of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA.

Primary aldosteronism (PA) is a common cause of secondary hypertension. Recent technological advances in genetic analysis have provided a better understanding of the molecular pathogenesis of this disease. The application of next-generation sequencing has resulted in the identification of somatic mutations in aldosterone-producing adenoma (APA), a major subtype of PA. Based on the recent findings using a sequencing method that selectively targets the tumor region where aldosterone synthase (CYP11B2) is expressed, the vast majority of APAs appear to harbor a somatic mutation in one of the aldosterone-driver genes, including KCNJ5, ATP1A1, ATP2B3, CACNA1D, CACNA1H, and CLCN2. Mutations in these genes alter intracellular ion homeostasis and enhance aldosterone production. In a small subset of APAs, somatic activating mutations in the CTNNB1 gene, which encodes β-catenin, have also been detected. Accumulating evidence suggests that race and sex impact the somatic mutation spectrum of APA. Specifically, somatic mutations in the KCNJ5 gene, encoding an inwardly rectifying K+ channel, are common in APAs from Asian populations as well as women regardless of race. Associations between APA histology, genotype, and patient clinical characteristics have also been proposed, suggesting a potential need to consider race and sex for the management of PA patients. Herein, we review recent findings regarding somatic mutations in APA and discuss potential roles of race and sex on the pathophysiology of APA as well as possible clinical implications.
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http://dx.doi.org/10.1530/EJE-21-0031DOI Listing
May 2021

Masking by hypokalemia-primary aldosteronism with undetectable aldosterone.

Clin Kidney J 2021 Apr 7;14(4):1269-1271. Epub 2020 Oct 7.

Stanford Hypertension Center, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.

Primary aldosteronism is the most common cause of secondary hypertension; however, the dynamic regulation of aldosterone by potassium is less well studied and current diagnostic recommendations are imprecise. We describe a young man who presented with resistant hypertension and severe hypokalemia. The workup initially revealed undetectable aldosterone despite acute potassium repletion. Chronic potassium supplementation eventually uncovered hyperaldosteronism. genetic studies revealed a gain-of-function mutation within an aldosterone-producing adenoma that was clinically responsive to changes in extracellular potassium. We highlight a unique presentation of Conn's syndrome and discuss the implications for the molecular mechanisms of potassium regulation of aldosterone.
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http://dx.doi.org/10.1093/ckj/sfaa150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8023175PMC
April 2021

Primary Cultures and Cell Lines for In Vitro Modeling of the Human Adrenal Cortex.

Tohoku J Exp Med 2021 04;253(4):217-232

Department of Molecular and Integrative Physiology, University of Michigan.

The human adrenal cortex is a complex endocrine organ that produces mineralocorticoids, glucocorticoids and androgens. These steroids are produced in distinct cell types located within the glomerulosa, fasciculata and reticularis of the adrenal cortex. Abnormal adrenal steroidogenesis leads to a variety of diseases that can cause hypertension, metabolic syndrome, infertility and premature adrenarche. The adrenal cortex can also develop steroid-producing adenomas and rarely adrenocortical carcinomas. In vitro cell culture models provide important tools to study molecular and cellular mechanisms controlling both the physiologic and pathologic conditions of the adrenal cortex. In addition, the presence of multiple steroid-metabolizing enzymes within adrenal cells makes it a model for defining possible endocrine disruptors that might block these enzymes. The regulation and dysregulation of human adrenal steroid production and cell division/tumor growth can be studied using freshly isolated cells but this requires access to human adrenal glands, which are not available to most investigators. Immortalized human adrenocortical cell lines have proven to be of considerable value in studying the molecular and biochemical mechanisms controlling adrenal steroidogenesis and tumorigenesis. Current human adrenal cell lines include the original NCI-H295 and its substrains: H295A, H295R, HAC13, HAC15, HAC50 and H295RA as well as the recently established MUC-1, CU-ACC1 and CU-ACC2. The current review will discuss the use of primary cultures of fetal and adult adrenal cells as well as adrenocortical cell lines as in vitro models for the study of human adrenal physiology and pathophysiology.
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http://dx.doi.org/10.1620/tjem.253.217DOI Listing
April 2021

Aldosterone-Regulating Receptors and Aldosterone-Driver Somatic Mutations.

Front Endocrinol (Lausanne) 2021 16;12:644382. Epub 2021 Mar 16.

Division of Metabolism, Endocrine, and Diabetes, University of Michigan, Ann Arbor, MI, United States.

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 (), ACTH receptors (), and melanocortin 2 receptor accessory protein () in APAs with known aldosterone-driver somatic mutations.

Methods: RNA was isolated from APAs with mutations in: (n = 14), (n = 14), (n = 14), and (n = 5), and from normal adjacent adrenal tissue (n = 45). Transcript expression of , , , aldosterone synthase (), 17α-hydroxylase/17,20-lyase (), and 11β-hydroxylase () were quantified using quantitative RT-PCR and normalized to β-actin.

Results: Compared to adjacent normal adrenal tissue, APAs had higher transcript levels of (2,216.4 [1,112.0, 2,813.5]-fold, < 0.001), (2.88 [2.00, 4.52]-fold,  < 0.001), and (1.80 [1.02, 2.80]-fold, < 0.001]), and lower transcript levels of , , and (0.28-0.36, < 0.001 for all). and transcripts were lower in APAs with other mutations ( < 0.01 for both). expression correlated positively with that of in APAs harboring and mutations, and with expression in APAs harboring ase mutations.

Conclusions: While and are expressed in all APAs, differences were observed based on the underlying aldosterone-driver somatic mutations. In tandem, our findings suggest that APAs with -mutations are more responsive to ACTH than -mutated APAs.
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http://dx.doi.org/10.3389/fendo.2021.644382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008747PMC
March 2021

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

J Clin Endocrinol Metab 2021 Apr;106(5):1389-1397

Division of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.

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), 3β-hydroxysteroid dehydrogenase type II (HSD3B2), 11β-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.
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http://dx.doi.org/10.1210/clinem/dgab007DOI Listing
April 2021

Corticotroph tumor progression after bilateral adrenalectomy (Nelson's syndrome): systematic review and expert consensus recommendations.

Eur J Endocrinol 2021 Mar;184(3):P1-P16

Section on Genetics & Endocrinology Eunice Kennedy Shriver National Insitute of Child Health & Human Development (NICHD) National Institute of Health (NIH), NIH Clinical Research Center, Bethesda, Maryland, USA.

Background: Corticotroph tumor progression (CTP) leading to Nelson's syndrome (NS) is a severe and difficult-to-treat complication subsequent to bilateral adrenalectomy (BADX) for Cushing's disease. Its characteristics are not well described, and consensus recommendations for diagnosis and treatment are missing.

Methods: A systematic literature search was performed focusing on clinical studies and case series (≥5 patients). Definition, cumulative incidence, treatment and long-term outcomes of CTP/NS after BADX were analyzed using descriptive statistics. The results were presented and discussed at an interdisciplinary consensus workshop attended by international pituitary experts in Munich on October 28, 2018.

Results: Data covered definition and cumulative incidence (34 studies, 1275 patients), surgical outcome (12 studies, 187 patients), outcome of radiation therapy (21 studies, 273 patients), and medical therapy (15 studies, 72 patients).

Conclusions: We endorse the definition of CTP-BADX/NS as radiological progression or new detection of a pituitary tumor on thin-section MRI. We recommend surveillance by MRI after 3 months and every 12 months for the first 3 years after BADX. Subsequently, we suggest clinical evaluation every 12 months and MRI at increasing intervals every 2-4 years (depending on ACTH and clinical parameters). We recommend pituitary surgery as first-line therapy in patients with CTP-BADX/NS. Surgery should be performed before extrasellar expansion of the tumor to obtain complete and long-term remission. Conventional radiotherapy or stereotactic radiosurgery should be utilized as second-line treatment for remnant tumor tissue showing extrasellar extension.
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http://dx.doi.org/10.1530/EJE-20-1088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8060870PMC
March 2021

Identification of Somatic Mutations in in Aldosterone-Producing Adenomas.

J Endocr Soc 2020 Oct 1;4(10):bvaa123. Epub 2020 Oct 1.

Division of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA.

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 in 2 APAs that were negative for currently known aldosterone-driver mutations. The gene encodes the voltage-gated chloride channel ClC-2. germline variants have previously been shown to cause familial hyperaldosteronism type II. Somatic mutations in were identified in 2 of 115 APAs, resulting in a prevalence of 1.74%. One of the 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 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.
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http://dx.doi.org/10.1210/jendso/bvaa123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7528565PMC
October 2020

Prevalence of Somatic Mutations in Aldosterone-Producing Adenomas in Japanese Patients.

J Clin Endocrinol Metab 2020 11;105(11)

Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan.

Context: Results of previous studies demonstrated clear racial differences in the prevalence of somatic mutations among patients with aldosterone-producing adenoma (APA). For instance, those in East Asian countries have a high prevalence of somatic mutations in KCNJ5, whereas somatic mutations in other aldosterone-driving genes are rare.

Objectives: To determine somatic mutation prevalence in Japanese APA patients using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided sequencing approach.

Method: Patients with a unilateral form of primary aldosteronism who underwent adrenalectomy at the Tohoku University Hospital were studied. Based on CYP11B2 immunolocalization of resected adrenals, genomic DNA was isolated from the relevant positive area of 10% formalin-fixed, paraffin-embedded tissue of the APAs. Somatic mutations in aldosterone-driving genes were studied in APAs by direct Sanger sequencing and targeted next-generation sequencing.

Results: CYP11B2 IHC-guided sequencing determined APA-related somatic mutations in 102 out of 106 APAs (96%). Somatic KCNJ5 mutation was the most frequent genetic alteration (73%) in this cohort of Japanese patients. Somatic mutations in other aldosterone-driving genes were also identified: CACNA1D (14%), ATP1A1 (5%), ATP2B3 (4%), and CACNA1H (1%), including 2 previously unreported mutations. KCNJ5 mutations were more often detected in APAs from female patients compared with those from male patients [95% (36/38) vs 60% (41/68); P < 0.0001].

Conclusion: IHC-guided sequencing defined somatic mutations in over 95% of Japanese APAs. While the dominance of KCNJ5 mutations in this particular cohort was confirmed, a significantly higher KCNJ5 prevalence was detected in female patients. This study provides a better understanding of genetic spectrum of Japanese APA patients.
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http://dx.doi.org/10.1210/clinem/dgaa595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7947976PMC
November 2020

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

Horm Metab Res 2020 Aug 13;52(8):607-613. Epub 2020 Aug 13.

Michigan Center for Translational Pathology, Ann Arbor, Michigan, USA.

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., , , 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.
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http://dx.doi.org/10.1055/a-1212-8803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880170PMC
August 2020

International Histopathology Consensus for Unilateral Primary Aldosteronism.

J Clin Endocrinol Metab 2021 Jan;106(1):42-54

Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Ludwig-Maximilians-Universität München, Germany.

Objective: Develop a consensus for the nomenclature and definition of adrenal histopathologic features in unilateral primary aldosteronism (PA).

Context: Unilateral PA is the most common surgically treated form of hypertension. Morphologic examination combined with CYP11B2 (aldosterone synthase) immunostaining reveals diverse histopathologic features of lesions in the resected adrenals.

Patients And Methods: Surgically removed adrenals (n = 37) from 90 patients operated from 2015 to 2018 in Munich, Germany, were selected to represent the broad histologic spectrum of unilateral PA. Five pathologists (Group 1 from Germany, Italy, and Japan) evaluated the histopathology of hematoxylin-eosin (HE) and CYP11B2 immunostained sections, and a consensus was established to define the identifiable features. The consensus was subsequently used by 6 additional pathologists (Group 2 from Australia, Brazil, Canada, Japan, United Kingdom, United States) for the assessment of all adrenals with disagreement for histopathologic diagnoses among group 1 pathologists.

Results: Consensus was achieved to define histopathologic features associated with PA. Use of CYP11B2 immunostaining resulted in a change of the original HE morphology-driven diagnosis in 5 (14%) of 37 cases. Using the consensus criteria, group 2 pathologists agreed for the evaluation of 11 of the 12 cases of disagreement among group 1 pathologists.

Conclusion: The HISTALDO (histopathology of primary aldosteronism) consensus is useful to standardize nomenclature and achieve consistency among pathologists for the histopathologic diagnosis of unilateral PA. CYP11B2 immunohistochemistry should be incorporated into the routine clinical diagnostic workup to localize the likely source of aldosterone production.
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http://dx.doi.org/10.1210/clinem/dgaa484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7765663PMC
January 2021

The Concordance Between Imaging and Adrenal Vein Sampling Varies With Aldosterone-Driver Somatic Mutation.

J Clin Endocrinol Metab 2020 10;105(10)

Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, Michigan.

Background: Correct subtyping of primary aldosteronism (PA) is critical for guiding clinical management. Adrenal imaging is less accurate than adrenal vein sampling (AVS); nonetheless, AVS is invasive, technically challenging, and scarcely available.

Objective: To identify predictors of concordance between cross-sectional imaging and lateralized AVS in patients with PA that could help circumvent AVS in a subset of patients.

Methods: We retrospectively studied all patients with PA who underwent AVS in a tertiary referral center from 2009 to 2019. AVS was performed before and after cosyntropin stimulation. Patients with lateralized AVS in at least one condition were included. Aldosterone synthase-guided next-generation sequencing was performed on available adrenal tissue. Logistic regression was implemented to identify predictors of imaging-AVS lateralization concordance.

Results: A total of 234 patients (62% men), age 20 to 79 years, 73% white, 23% black, and 2% Asian were included. AVS lateralization was found: 1) both pre- and post-cosyntropin (Uni/Uni) in 138 patients; 2) only at baseline (Uni/Bi) in 39 patients; 3) only after cosyntropin stimulation (Bi/Uni) in 29 patients. Catheterization partially failed in 28 patients. AVS-imaging agreement was higher in patients with KCNJ5 versus other aldosterone-driver somatic mutations (90.3% versus 64.6%; P < 0.001); in Asian and white versus black Americans (75%, 70%, and 36%, respectively); in younger patients; and those with left adrenal nodules and contralateral suppression. Conversely, AVS-imaging agreement was lowest in Uni/Bi patients (38% vs. 69% in Uni/Uni, and 62% in Bi/Uni; P = 0.007).

Conclusions: While AVS-imaging agreement is higher in young white and Asian patients, who have KCNJ5-mutated aldosterone producing adenomas, no predictor confers absolute imaging accuracy.
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http://dx.doi.org/10.1210/clinem/dgaa482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437239PMC
October 2020

Biochemical, Histopathological, and Genetic Characterization of Posture-Responsive and Unresponsive APAs.

J Clin Endocrinol Metab 2020 09;105(9)

Endocrine Hypertension Research Centre, University of Queensland Diamantina Institute, Greenslopes and Princess Alexandra Hospitals, Brisbane, Australia.

Context And Objective: Posture-responsive and posture-unresponsive aldosterone-producing adenomas (APAs) account for approximately 40% and 60% of APAs, respectively. Somatic gene mutations have been recently reported to exist in approximately 90% of APAs. This study was designed to characterize the biochemical, histopathologic, and genetic properties of these 2 types of APA.

Methods: Plasma levels of aldosterone and hybrid steroids (18-oxocortisol and 18-hydroxycortisol) were measured by liquid chromatography-tandem mass spectrometry. Immunohistochemistry for CYP11B2 (aldosterone synthase) and CYP17A1 (17α-hydroxylase) and deoxyribonucleic acid sequencing (Sanger and next-generation sequencing) were performed on APA tissue collected from 23 posture-unresponsive and 17 posture-responsive APA patients.

Results: Patients with posture-unresponsive APA displayed higher (P < 0.01) levels of hybrid steroids, recumbent aldosterone and cortisol, larger (P < 0.01) zona fasciculata (ZF)-like tumors with higher (P < 0.01) expression of CYP17A1 (but not of CYP11B2) than patients with posture-responsive APA (most of which were not ZF-like). Of 40 studied APAs, 37 (92.5%) were found to harbor aldosterone-driving somatic mutations (KCNJ5 = 14 [35.0%], CACNA1D = 13 [32.5%], ATP1A1 = 8 [20.0%], and ATP2B3 = 2 [5.0%]), including 5 previously unreported mutations (3 in CACNA1D and 2 in ATP1A1). Notably, 64.7% (11/17) of posture-responsive APAs carried CACNA1D mutations, whereas 56.5% (13/23) of posture-unresponsive APAs harbored KCNJ5 mutations.

Conclusions: The elevated production of hybrid steroids by posture-unresponsive APAs may relate to their ZF-like tumor cell composition, resulting in expression of CYP17A1 (in addition to somatic gene mutation-driven CYP11B2 expression), thereby allowing production of cortisol, which acts as the substrate for CYP11B2-generated hybrid steroids.
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http://dx.doi.org/10.1210/clinem/dgaa367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426003PMC
September 2020

Sex Differences in 11-Oxygenated Androgen Patterns Across Adulthood.

J Clin Endocrinol Metab 2020 08;105(8)

Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, Ann Arbor, MI, US.

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), 11β-hydroxytestosterone (11OHT), 11-ketotestosterone (11KT), 11β-hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), cortisol, and cortisone in morning sera obtained from adults in outpatient setting. We performed double immunofluorescence of 3β-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 17β-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.
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http://dx.doi.org/10.1210/clinem/dgaa343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340191PMC
August 2020

The Potential Role of Aldosterone-Producing Cell Clusters in Adrenal Disease.

Horm Metab Res 2020 Jun 30;52(6):427-434. Epub 2020 Mar 30.

Departments of Molecular, Integrative Physiology and Internal Medicine, University of Michigan, Ann Arbor, MI, USA.

Primary aldosteronism (PA) is the most common cause of secondary hypertension. The hallmark of PA is adrenal production of aldosterone under suppressed renin conditions. PA subtypes include adrenal unilateral and bilateral hyperaldosteronism. Considerable progress has been made in defining the role for somatic gene mutations in aldosterone-producing adenomas (APA) as the primary cause of unilateral PA. This includes the use of next-generation sequencing (NGS) to define recurrent somatic mutations in APA that disrupt calcium signaling, increase aldosterone synthase (CYP11B2) expression, and aldosterone production. The use of CYP11B2 immunohistochemistry on adrenal glands from normal subjects, patients with unilateral and bilateral PA has allowed the identification of CYP11B2-positive cell foci, termed aldosterone-producing cell clusters (APCC). APCC lie beneath the adrenal capsule and like APA, many APCC harbor somatic gene mutations known to increase aldosterone production. These findings suggest that APCC may play a role in pathologic progression of PA. Herein, we provide an update on recent research directed at characterizing APCC and also discuss the unanswered questions related to the role of APCC in PA.
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http://dx.doi.org/10.1055/a-1128-0421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8029307PMC
June 2020

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

Gland Surg 2020 Feb;9(1):3-13

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA.

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 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 mutations. Herein, we review the recent advances with respect to the molecular mechanisms underlying the pathogenesis of PA and the steroidogenic fingerprints of mutations. In addition, we present an outlook toward the future of PA subtyping and diagnostic work-up utilizing steroid profiling.
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http://dx.doi.org/10.21037/gs.2019.10.22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082274PMC
February 2020

11-Oxygenated androgens in health and disease.

Nat Rev Endocrinol 2020 05 16;16(5):284-296. Epub 2020 Mar 16.

Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, Ann Arbor, MI, USA.

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 11β-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.
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http://dx.doi.org/10.1038/s41574-020-0336-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881526PMC
May 2020

Genetic, Cellular, and Molecular Heterogeneity in Adrenals With Aldosterone-Producing Adenoma.

Hypertension 2020 04 2;75(4):1034-1044. Epub 2020 Mar 2.

From the PARCC, INSERM, Université de Paris, France (K.D.S., S. Boulkroun, A.R., I.G.-D., L.A., F.L.F.-R., M.-C.Z.).

Aldosterone-producing adenoma (APA) cause primary aldosteronism-the most frequent form of secondary hypertension. Somatic mutations in genes coding for ion channels and ATPases are found in APA and in aldosterone-producing cell clusters. We investigated the genetic, cellular, and molecular heterogeneity of different aldosterone-producing structures in adrenals with APA, to get insight into the mechanisms driving their development and to investigate their clinical and biochemical correlates. Genetic analysis of APA, aldosterone-producing cell clusters, and secondary nodules was performed in adrenal tissues from 49 patients by next-generation sequencing following CYP11B2 immunohistochemistry. Results were correlated with clinical and biochemical characteristics of patients, steroid profiles, and histological features of the tumor and adjacent adrenal cortex. Somatic mutations were identified in 93.75% of APAs. Adenoma carrying mutations had more clear cells and cells expressing CYP11B1, and fewer cells expressing CYP11B2 or activated β-catenin, compared with other mutational groups. 18-hydroxycortisol and 18-oxocortisol were higher in patients carrying mutations and correlated with histological features of adenoma; however, mutational status could not be predicted using steroid profiling. Heterogeneous CYP11B2 expression in -mutated adenoma was not associated with genetic heterogeneity. Different mutations were identified in secondary nodules expressing aldosterone synthase and in independent aldosterone-producing cell clusters from adrenals with adenoma; known mutations were identified in 5 aldosterone-producing cell clusters. Genetic heterogeneity in different aldosterone-producing structures in the same adrenal suggests complex mechanisms underlying APA development.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.119.14177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098445PMC
April 2020

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

Horm Cancer 2020 02 30;11(1):52-62. Epub 2020 Jan 30.

Department of Internal Medicine, Division of Metabolism, Endocrinology & Diabetes, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, MI, 48109, USA.

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.
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http://dx.doi.org/10.1007/s12672-019-00375-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018534PMC
February 2020

Somatic Mutation As a Cause of Aldosterone-Producing Adenoma.

Hypertension 2020 03 27;75(3):645-649. Epub 2020 Jan 27.

From the Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor (K.N., A.R.B., J.R., W.E.R.).

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 mutation (c.T4289C, p.I1430T) in an APA without any currently known aldosterone-driver mutations using CYP11B2 immunohistochemistry-guided whole exome sequencing. The 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 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 mRNA expression using the human adrenocortical HAC15 cell line with a doxycycline-inducible mutation. Doxycycline treatment increased mRNA levels as well as aldosterone production, supporting a pathological role of the p.I1430T mutation on the development of primary aldosteronism. In conclusion, somatic 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.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.119.14349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059016PMC
March 2020

Histological Characterization of Aldosterone-producing Adrenocortical Adenomas with Different Somatic Mutations.

J Clin Endocrinol Metab 2020 03;105(3)

Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan.

Context: Aldosterone-producing adrenocortical adenomas (APAs) are mainly composed of clear (lipid rich) and compact (eosinophilic) tumor cells. The detailed association between these histological features and somatic mutations (KCNJ5, ATP1A1, ATP2B3, and CACNA1D) in APAs is unknown.

Objective: To examine the association between histological features and individual genotypes in APAs.

Methods: Examination of 39 APAs subjected to targeted next-generation sequencing (11 KCNJ5, 10 ATP1A1, 10 ATP2B3, and 8 CACNA1D) and quantitative morphological and immunohistochemical (CYP11B2 and CYP17A1) analyses using digital imaging software.

Results: KCNJ5- and ATP2B3-mutated APAs had clear cell dominant features (KCNJ5: clear 59.8% [54.4-64.6%] vs compact 40.2% (35.4-45.6%), P = .0022; ATP2B3: clear 54.3% [48.2-62.4 %] vs compact 45.7% (37.6-51.8 %), P = .0696). ATP1A1- and CACNA1D-mutated APAs presented with marked intratumoral heterogeneity. A significantly positive correlation of immunoreactivity was detected between CYP11B2 and CYP17A1 in tumor cells of KCNJ5-mutated APAs (P = .0112; ρ = 0.7237), in contrast, significantly inverse correlation was detected in ATP1A1-mutated APAs (P = .0025; ρ = -0.8667).

Conclusion: KCNJ5-mutated APAs, coexpressing CYP11B2 and CYP17A1, were more deviated in terms of zonation-specific differentiation of adrenocortical cells than ATP1A1- and ATP2B3-mutated APAs.
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http://dx.doi.org/10.1210/clinem/dgz235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048684PMC
March 2020

Comprehensive Analysis of Steroid Biomarkers for Guiding Primary Aldosteronism Subtyping.

Hypertension 2020 01 2;75(1):183-192. Epub 2019 Dec 2.

From the Division of Metabolism, Endocrinology, and Diabetes (A.F.T., T.W., A.T.N., J.R., P.J.O., W.E.R., R.J.A.), University of Michigan, Ann Arbor.

Adrenal vein sampling (AVS) is required to distinguish unilateral from bilateral aldosterone sources in primary aldosteronism (PA), and cortisol is used for AVS data interpretation, but cortisol has several pitfalls. In this study, we present the utility of several other steroids in PA subtyping, both during AVS, as well as in peripheral serum. We included patients with PA who underwent AVS at University of Michigan between 2012 and 2018. We used mass spectrometry to simultaneously quantify 17 steroids in adrenal veins (AV) and periphery, both at baseline and after cosyntropin administration. PA was classified as unilateral or bilateral based on a lateralization index ≥ or <4, respectively, separately for baseline and post-cosyntropin administration. Of 131 participants, AV catheterizations was deemed failed in 28 (21 %) patients (36 AVs) at baseline. Eight steroids demonstrated higher AV/periphery ratios than cortisol (<0.01 for all); 11β-hydroxyandrostenedione, 11-deoxycortisol, and corticosterone rescued most failed baseline catheterizations. Lateralization was generally consistent when using these alternative steroids. Based on pre- and post-cosyntropin data, the remaining 103 patients were classified as: U/U, 37; B/B, 32; U/B, 20; B/U, 14. Discriminant analysis of multi-steroid panels from peripheral serum showed distinct profiles across the 4 groups, with highest aldosterone, 18-oxocortisol and 11-deoxycorticosterone in U/U patients. In conclusion, 11β-hydroxyandrostenedione and 11-deoxycortisol are superior to cortisol for AVS data interpretation. Single assay multi-steroid panels measured in peripheral serum are helpful in stratified PA subtyping and have the potential to circumvent AVS in a subset of patients with PA.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.119.13866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034384PMC
January 2020

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

J Clin Invest 2020 01;130(1):83-93

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA.

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.
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http://dx.doi.org/10.1172/JCI127429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934213PMC
January 2020

Three Discrete Patterns of Primary Aldosteronism Lateralization in Response to Cosyntropin During Adrenal Vein Sampling.

J Clin Endocrinol Metab 2019 12;104(12):5867-5876

Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, Ann Arbor, Michigan.

Context: Cosyntropin [ACTH (1-24)] stimulation during adrenal vein (AV) sampling (AVS) enhances the confidence in the success of AV cannulation and circumvents intraprocedure hormonal fluctuations. Cosyntropin's effect on primary aldosteronism (PA) lateralization, however, is controversial.

Objectives: To define the major patterns of time-dependent lateralization, and their determinants, after cosyntropin stimulation during AVS.

Methods: We retrospectively studied patients with PA who underwent AVS before, 10, and 20 minutes after cosyntropin stimulation between 2009 and 2018. Unilateral (U) or bilateral (B) PA was determined on the basis of a lateralization index (LI) value ≥4 or <4, respectively. Available adrenal tissue underwent aldosterone synthase-guided next-generation sequencing.

Results: PA lateralization was concordant between basal and cosyntropin-stimulated AVS in 169 of 222 patients (76%; U/U, n = 110; B/B, n = 59) and discordant in 53 patients (24%; U/B, n = 32; B/U, n = 21). Peripheral and dominant AV aldosterone concentrations and LI were highest in U/U patients and progressively lower across intermediate and B/B groups. LI response to cosyntropin increased in 27% of patients, decreased in 33%, and remained stable in 40%. Baseline aldosterone concentrations predicted the LI pattern across time (P < 0.001). Mutation status was defined in 61 patients. Most patients with KCNJ5 mutations had descending LI, whereas those with ATP1A1 and ATP2B3 mutations had ascending LI after cosyntropin stimulation.

Conclusion: Patients with severe PA lateralized robustly regardless of cosyntropin use. Cosyntropin stimulation reveals intermediate PA subtypes; its impact on LI varies with baseline aldosterone concentrations and aldosterone-driver mutations.
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http://dx.doi.org/10.1210/jc.2019-01182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800532PMC
December 2019
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