Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands

Abstract

Primary aldosteronism (PA) represents the most common cause of secondary hypertension, but little is known regarding its adrenal cellular origins. Recently, aldosterone-producing cell clusters (APCCs) with high expression of aldosterone synthase (CYP11B2) were found in both normal and PA adrenal tissue. PA-causing aldosterone-producing adenomas (APAs) harbor mutations in genes encoding ion channels/pumps that alter intracellular calcium homeostasis and cause renin-independent aldosterone production through increased CYP11B2 expression. Herein, we hypothesized that APCCs have APA-related aldosterone-stimulating somatic gene mutations. APCCs were studied in 42 normal adrenals from kidney donors. To clarify APCC molecular characteristics, we used microarrays to compare the APCC transcriptome with conventional adrenocortical zones [zona glomerulosa (ZG), zona fasciculata, and zona reticularis]. The APCC transcriptome was most similar to ZG but with an enhanced capacity to produce aldosterone. To determine if APCCs harbored APA-related mutations, we performed targeted next generation sequencing of DNA from 23 APCCs and adjacent normal adrenal tissue isolated from both formalin-fixed, paraffin-embedded, and frozen tissues. Known aldosterone driver mutations were identified in 8 of 23 (35%) APCCs, including mutations in calcium channel, voltage-dependent, L-type, α1D-subunit (CACNA1D; 6 of 23 APCCs) and ATPase, Na(+)/(K+) transporting, α1-polypeptide (ATP1A1; 2 of 23 APCCs), which were not observed in the adjacent normal adrenal tissue. Overall, we show three major findings: (i) APCCs are common in normal adrenals, (ii) APCCs harbor somatic mutations known to cause excess aldosterone production, and (iii) the mutation spectrum of aldosterone-driving mutations is different in APCCs from that seen in APA. These results provide molecular support for APCC as a precursor of PA.

Keywords: adrenal; aldosterone; aldosterone-producing cell cluster; primary aldosteronism; somatic mutations.

Fig. 1.

APCC transcriptome comparison with adrenal ZG, ZF, and ZR. (A) Principal component analyses using microarray analysis after estimated subject effects were removed for four adrenal cell populations. Log₂-transformed values are used for the graphs. PC, principal component. (B) Heat map of genes with a mean expression variation of greater than threefold between APCC and ZG (P < 0.01). Only probe sets annotated as representing a known gene are shown, and the probe set for each gene shown is the one with largest APCC vs. ZG fold change. (C) qPCR analysis of CYP11B2 in four adrenocortical tissues (APCC/ZG/ZF/ZR) from four subjects. P values are from two-way ANOVA models with terms for subjects and tissues. Error bars are SEMs.

Fig. S1.

Principal component analysis of the microarray. Plots of the first three principal components [PC #1–3 (#1 vs. #2 for A and #1 vs. #3 for B)] using log₂-transformed data for all probe sets. Subject numbers (DAN sample numbers) are labeled to show that PC3 indicates important subject effects (individual adrenals), with DAN samples 45 and 46 having greater PC3 than DAN samples 48 and 50 within every tissue.

Fig. 2.

APCC score (frequency and size) during aging. (A) CYP11B2 immunohistochemistry from a normal adrenal (DAN22) showing an example of small APCCs (blue arrows). (B) CYP11B2 immunohistochemistry for DAN11 with examples of large APCCs (red arrows). (C) Scatter plot of average APCC score (from five observers) vs. age and sex of patient.

Fig. 3.

Identification of APCCs in FFPE tissues for targeted NGS (only mutated samples are shown). Six consecutive 5-µm FFPE sections were cut from blocks containing histologically benign adrenal glands. APCCs were identified after CYP11B2 immunohistochemistry of the first and last sections. Careful macrodissection was performed on intervening sections to isolate APCCs or adjacent normal adrenal tissue. Boxed areas indicate the APCC or normal tissue regions that were isolated. For each case with an identified somatic mutation (Table 2), APCCs and normal adrenal tissue subjected to sequencing are indicated. (Scale bar: 1 cm.)

Fig. 4.

LCM of APCC for targeted NGS. APCCs from DAN45 and -50 were identified by CYP11B2 immunostaining on frozen tissue sections for NGS. (A) Low-magnification view of DAN50 with the regions captured for APCC 50 (red box) and paired normal adrenal tissue (yellow box) shown. (B–E) Photomicrographs showing (B and D) pre- and (C and E) post-LCM images that confirm isolation of desired cell populations. A somatic CACNA1D F747L mutation was identified exclusively in the APCC component (Table 2).

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