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

ABSTRACT: 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 KCNJ5 mutations had more clear cells and cells expressing CYP11B1, and fewer cells expressing CYP11B2 or activated ?-catenin, compared with other mutational groups. 18-hydroxycortisol and 18-oxocortisol were higher in patients carrying KCNJ5 mutations and correlated with histological features of adenoma; however, mutational status could not be predicted using steroid profiling. Heterogeneous CYP11B2 expression in KCNJ5-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 KCNJ5 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.

SUBMITTER: De Sousa K

PROVIDER: S-EPMC7098445 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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

De Sousa Kelly K Boulkroun Sheerazed S Baron Stéphanie S Nanba Kazutaka K Wack Maxime M Rainey William E WE Rocha Angélique A Giscos-Douriez Isabelle I Meatchi Tchao T Amar Laurence L Travers Simon S Fernandes-Rosa Fabio L FL Zennaro Maria-Christina MC

Hypertension (Dallas, Tex. : 1979) 20200302 4

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. Gene ...[more]

PMID: 32114847

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Project description:ContextThe use of next-generation sequencing has resulted in the identification of recurrent somatic mutations underlying primary aldosteronism (PA). However, significant gaps remain in our understanding of the relationship between tumor aldosterone synthase (CYP11B2) expression and somatic mutation status.ObjectiveThe objective of the study was to investigate tumor CYP11B2 expression and somatic aldosterone-driver gene mutation heterogeneity.MethodsFifty-one adrenals from 51 PA patients were studied. Immunohistochemistry for CYP11B2 was performed. Aldosterone-producing adenomas with intratumor CYP11B2 heterogeneity were analyzed for mutation status using targeted next-generation sequencing. DNA was isolated from CYP11B2-positive, CYP11B2-negative, and adjacent normal areas from formalin-fixed, paraffin-embedded sections.ResultsOf 51 adrenals, seven (14 %) showed distinct heterogeneity in CYP11B2 by immunohistochemistry, including six adenomas with intratumor heterogeneity and one multinodular hyperplastic adrenal with both CYP11B2-positive and -negative nodules. Of the six adrenocortical adenomas with CYP11B2 heterogeneity, three had aldosterone-regulating mutations (CACNA1D p.F747C, KCNJ5 p.L168R, ATP1A1 p.L104R) only in CYP11B2-positive regions, and one had two different mutations localized to two histologically distinct CYP11B2-positive regions (ATP2B3 p.L424_V425del, KCNJ5 p.G151R). Lastly, one adrenal with multiple CYP11B2-expressing nodules showed different mutations in each (CACNA1D p.F747V and ATP1A1 p.L104R), and no mutations were identified in CYP11B2-negative nodule or adjacent normal adrenal.ConclusionsAdrenal tumors in patients with PA can demonstrate clear heterogeneity in CYP11B2 expression and somatic mutations in driver genes for aldosterone production. These findings suggest that aldosterone-producing adenoma tumorigenesis can occur within preexisting nodules through the acquisition of somatic mutations that drive aldosterone production.

Project description:The endoplasmic reticulum (ER) plays a pivotal role in syntheses of proteins and steroid hormones and regulation of intracellular Ca2+ level. We aimed to investigate ER-associated genes in aldosterone-producing adenomas (APAs) and clarify their effect on aldosterone production. Microarray analysis targeting 288 ER-associated genes was conducted using nonfunctioning adrenocortical adenomas (n=5) and APAs (n=19). Immunohistochemistry and quantitative polymerase chain reaction analyses were performed with 13 nonfunctioning adrenocortical adenoma and 48 APA samples. Functional studies were performed with human adrenocortical carcinoma (HAC15) cells, some of which were genetically modified using lentiviruses. The ER chaperone calmegin (CLGN) was the most highly expressed ER-associated gene in APAs relative to nonfunctioning adrenocortical adenomas. Analysis with quantitative polymerase chain reaction revealed CLGN to be 9.5-fold upregulated in APAs relative to nonfunctioning adrenocortical adenomas. There were no differences among different APA genotypes affecting aldosterone production. Immunohistochemistry analysis revealed that CLGN was strongly expressed in APAs and aldosterone-producing cell clusters. Angiotensin II stimulation or KCNJ5 T158A overexpression in HAC15 cells did not affect CLGN mRNA levels. CLGN overexpression in HAC15 cells increased aldosterone levels but did not stimulate CYP11B2 mRNA levels. Pathway and gene ontology analyses using RNA sequencing results showed that tRNA aminoacyl metabolism was the most enriched pathway in CLGN-overexpressing cells. CYP11B2 (aldosterone synthase) and HSD3B2 (3 beta-hydroxysteroid dehydrogenase/delta 5->4-isomerase type 2) protein expression were more abundant in CLGN-overexpressing cells. CLGN knockdown using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) method in HAC15 cells that carry the KCNJ5 mutation did not affect aldosterone production. To summarize, CLGN was upregulated and associated with aldosterone production via translational regulation of CYP11B2 in APAs.

Dataset Information

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

Publications

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

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