*Case Report* **Familial Hyperaldosteronism Type 3 with a Rapidly Growing Adrenal Tumor: An In Situ Aldosterone Imaging Study**

**Nae Takizawa 1,† , Susumu Tanaka 2,† , Koshiro Nishimoto 3,\* ,†, Yuki Sugiura <sup>4</sup> , Makoto Suematsu <sup>4</sup> , Chisato Ohe <sup>5</sup> , Haruyuki Ohsugi <sup>1</sup> , Yosuke Mizuno <sup>6</sup> , Kuniaki Mukai <sup>4</sup> , Tsugio Seki <sup>7</sup> , Kenji Oki <sup>8</sup> , Celso E. Gomez-Sanchez <sup>9</sup> and Tadashi Matsuda <sup>1</sup>**


**Abstract:** Primary aldosteronism is most often caused by aldosterone-producing adenoma (APA) and bi-lateral adrenal hyperplasia. Most APAs are caused by somatic mutations of various ion channels and pumps, the most common being the inward-rectifying potassium channel *KCNJ5*. Germ line mutations of *KCNJ5* cause familial hyperaldosteronism type 3 (FH3), which is associated with severe hyperaldosteronism and hypertension. We present an unusual case of FH3 in a young woman, first diagnosed with primary aldosteronism at the age of 6 years, with bilateral adrenal hyperplasia, who underwent unilateral adrenalectomy (left adrenal) to alleviate hyperaldosteronism. However, her hyperaldosteronism persisted. At the age of 26 years, tomography of the remaining adrenal revealed two different adrenal tumors, one of which grew substantially in 4 months; therefore, the adrenal gland was removed. A comprehensive histological, immunohistochemical, and molecular evaluation of various sections of the adrenal gland and in situ visualization of aldosterone, using matrix-assisted laser desorption/ionization imaging mass spectrometry, was performed. Aldosterone synthase (CYP11B2) immunoreactivity was observed in the tumors and adrenal gland. The larger tumor also harbored a somatic β-catenin activating mutation. Aldosterone visualized in situ was only found in the subcapsular regions of the adrenal and not in the tumors. Collectively, this case of FH3 presented unusual tumor development and histological/molecular findings.

**Keywords:** familial hyperaldosteronism type 3; *KCNJ5*; adrenal tumor; β-catenin; MALDI-IMS; CYP11B2

#### **1. Introduction**

Primary aldosteronism (PA) is caused by excessive and autonomous secretion of aldosterone and is classified with aldosterone-producing adenoma (APA), bilateral idiopathic hyperaldosteronism (IHA), unilateral hyperplasia, or aldosterone-producing carcinoma.

**Citation:** Takizawa, N.; Tanaka, S.; Nishimoto, K.; Sugiura, Y.; Suematsu, M.; Ohe, C.; Ohsugi, H.; Mizuno, Y.; Mukai, K.; Seki, T.; et al. Familial Hyperaldosteronism Type 3 with a Rapidly Growing Adrenal Tumor: An In Situ Aldosterone Imaging Study. *Curr. Issues Mol. Biol.* **2022**, *44*, 128–138. https://doi.org/10.3390/ cimb44010010

Academic Editor: Dumitru A. Iacobas

Received: 22 November 2021 Accepted: 21 December 2021 Published: 28 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Somatic mutations in ion channel/pump genes, including the inwardly rectifying subfamily J, member 5 potassium channel (*KCNJ5*), have been identified in a significant percentage of APAs (APA-associated mutations) [1]. *KCNJ5* mutations cause a loss in specificity of the channel's selectivity filter for potassium. This leads to sodium leakage into the cells, causing depolarization of the membrane potential; this results in increased calcium influx into adrenocortical cells, causing autonomous aldosterone production [1]. There are four types of familial hyperaldosteronism (FH1–FH4) [2]. FH3 is caused by a germline mutation of *KCNJ5* that leads to adrenal hyperplasia with a marked increase in the secretion of aldosterone [1]. We and others have recently reported cases of non-familial juvenile PA due to mosaicism of somatic *KCNJ5*-mutated and non-mutated cells [3,4], in which the mutated cells/tissues were hyperplastic.

We previously described an immunohistochemistry protocol for aldosterone synthase (CYP11B2) that distinguishes CYP11B2 from the cortisol-synthesizing enzyme steroid 11βhydroxylase (CYP11B1) [5]. Using CYP11B2 staining, putative aldosterone-producing cells were visualized in the zona glomerulosa of normal adrenals from infants and adults [5–7], as well as several PA lesions [4,5,8]. However, aldosterone biosynthesis requires a cascade of steroidogenic enzymes, and the presence of CYP11B2 alone is not sufficient for the synthesis of aldosterone. To visualize the aldosterone localization in adrenal sections, we recently developed a protocol for the in situ detection of aldosterone using state-of-the-art matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) [9]. Since the steroid hormones, including aldosterone, are released into the blood stream immediately after production, i.e., there is no intracellular storage of steroid hormones, the detection of aldosterone in cells using MALDI-IMS indicates that those cells are actively producing aldosterone. In the present study, we describe an FH3 case with results of comprehensive molecular and CYP11B2 immunohistochemical analyses and correlated them with aldosterone localization in adrenal tissue.

#### **2. Case**

We present the case of a 27-year-old Japanese female with a history of severe juvenile PA. She was diagnosed with PA due to bilateral adrenal hyperplasia following adrenal vein sampling at the age of six years and treated with spironolactone and potassium supplementation with moderate control of her blood pressure [10]. However, when she was 15 years old, her serum creatinine level increased to 2.04 mg/dL (normal range: 0.4–1 mg/dL) due to severe hypertension and persistent high plasma aldosterone concentration (PAC: 2511 pg/mL (normal range: 35.7–240 pg/mL)). Since multiple adrenal vein catheterization attempts failed, and computed tomography (CT) indicated that her left adrenal gland (pink arrowhead in Figure 1A) was more hyperplastic than the right, she underwent left adrenalectomy, expecting to alleviate hyperaldosteronism. However, although lower than before, PAC remained elevated (1280 pg/mL) after surgery. At the age of 21 years, she developed end-stage chronic renal failure, thereby requiring intermittent hemodialysis. When she was 26 years old, a CT detected two adrenal tumors (22 × 17 mm and 10 × 6 mm) in her right adrenal gland (red and blue arrowheads in Figure 1B, respectively). Four months later, the larger tumor grew further (28 × 25 mm), and the smaller tumor remained unchanged (Figure 1C), suggesting that the larger tumor might be an adrenocortical carcinoma. She underwent a laparoscopic right adrenalectomy with removal of the intact gland in toto. Her PAC fell into the normal range (83 pg/mL) while on replacement with prednisolone for bilateral adrenalectomy.

**Figure 1.** CT and histological findings of the case. (**A**) CT findings at 15 years of age. The left adrenal gland was removed after CT examination. (**B**) CT findings at 26 years of age. Red and blue arrowheads indicate the larger and smaller adrenal tumors in the right adrenal gland, respectively. (**C**) CT findings 4 months after the CT shown in panel B. The larger tumor significantly enlarged in 4 months. (**D**) Macroscopic findings of the extracted right adrenal. The larger (\*) and the smaller (#) tumors presumably corresponded to the large (red arrowhead) and small (blue arrowhead) tumors in panels (B) and (**C**), respectively. The adrenal was cut into 16 pieces at the green lines. (**E**) Cut surfaces of the extracted adrenal. The green numbers in panel (**D**) correspond to the numbers in panel (**E**). The cut surface numbers in panels (**D**,**E**) correspond to those in parentheses in Supplementary Figure S1, which shows the sections after formaldehyde fixation. Frozen tissue blocks, in an optimal cutting temperature compound, were prepared from 4 portions, indicated by white frames (FB5, FB10, FB15- 1, and FB15-2). Flash frozen tissues were also taken from 3 non-tumor portions (N1–N3) and 3 tumor portions (T1–T3). (**F**–**I**) Hematoxylin and eosin staining, immunohistochemistry for CYP11B2, MALDI-imaging of aldosterone and cortisone (aldo/cortisone), and that of 18-oxocortisol (18oxoF), respectively, of frozen tissues. **3. Materials and Methods**  *3.1. DNA and RNA Isolation from Flash Frozen Tissues, Blood, and Hair Root*  Using the AllPrep DNA/RNA Mini Kit (catalog#: 80204, Qiagen, Valencia, CA, USA) and ISOHAIR (catalog#: 315-3403, NIPPON GENE CO., LTD., Tokyo, Japan), genomic **Figure 1.** CT and histological findings of the case. (**A**) CT findings at 15 years of age. The left adrenal gland was removed after CT examination. (**B**) CT findings at 26 years of age. Red and blue arrowheads indicate the larger and smaller adrenal tumors in the right adrenal gland, respectively. (**C**) CT findings 4 months after the CT shown in panel B. The larger tumor significantly enlarged in 4 months. (**D**) Macroscopic findings of the extracted right adrenal. The larger (\*) and the smaller (#) tumors presumably corresponded to the large (red arrowhead) and small (blue arrowhead) tumors in panels (**B**) and (**C**), respectively. The adrenal was cut into 16 pieces at the green lines. (**E**) Cut surfaces of the extracted adrenal. The green numbers in panel (**D**) correspond to the numbers in panel (**E**). The cut surface numbers in panels (**D**,**E**) correspond to those in parentheses in Supplementary Figure S1, which shows the sections after formaldehyde fixation. Frozen tissue blocks, in an optimal cutting temperature compound, were prepared from 4 portions, indicated by white frames (FB5, FB10, FB15-1, and FB15-2). Flash frozen tissues were also taken from 3 non-tumor portions (N1–N3) and 3 tumor portions (T1–T3). (**F**–**I**) Hematoxylin and eosin staining, immunohistochemistry for CYP11B2, MALDI-imaging of aldosterone and cortisone (aldo/cortisone), and that of 18-oxocortisol (18oxoF), respectively, of frozen tissues.

#### DNA and RNA (DNA/RNA) #86, 87, 88, 89, 90, and 91 were prepared from N1, N2, N3, T1, T2, and T3, respectively, as previously reported [11]. DNA #92, 93, 155, and 156 were **3. Materials and Methods**

#### isolated from the patient's blood, mother's blood, patient's hair root, and father's blood, *3.1. DNA and RNA Isolation from Flash Frozen Tissues, Blood, and Hair Root*

respectively, according to the manufacturer's instruction. *3.2. Whole Exome Sequencing*  We performed whole exome sequencing of genomic DNA samples from T1, N1, and blood (Bl), which was carried out at RIKEN GENESIS CO., LTD. (Tokyo, Japan), as follows. DNA was sheared into approximately 200 bp fragments and used to construct a Using the AllPrep DNA/RNA Mini Kit (catalog#: 80204, Qiagen, Valencia, CA, USA) and ISOHAIR (catalog#: 315-3403, NIPPON GENE CO., LTD., Tokyo, Japan), genomic DNA and RNA (DNA/RNA) #86, 87, 88, 89, 90, and 91 were prepared from N1, N2, N3, T1, T2, and T3, respectively, as previously reported [11]. DNA #92, 93, 155, and 156 were isolated from the patient's blood, mother's blood, patient's hair root, and father's blood, respectively, according to the manufacturer's instruction.

#### log#: G9641B, Agilent Technologies, Santa Clara, CA, USA). The constructed library was *3.2. Whole Exome Sequencing*

We performed whole exome sequencing of genomic DNA samples from T1, N1, and blood (Bl), which was carried out at RIKEN GENESIS CO., LTD. (Tokyo, Japan), as follows.

library for multiplexed paired-end sequencing with the SureSelectXT Reagent Kit (cata-

DNA was sheared into approximately 200 bp fragments and used to construct a library for multiplexed paired-end sequencing with the SureSelectXT Reagent Kit (catalog#: G9641B, Agilent Technologies, Santa Clara, CA, USA). The constructed library was hybridized to biotinylated cRNA baits from the SureSelectXT Human All Exon V6 Kit (catalog#: 5190–8865, Agilent Technologies, Santa Clara, CA, USA) for target enrichment. Targeted sequences were purified with magnetic beads, amplified, and sequenced on an Illumina HiSeq 2500 platform in paired-end 101 bp configuration.

The raw sequence read data of the three samples passed the quality checks in FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on 1 February 2017)). Read trimming via base quality was performed using Trimmomatic [12]. Read alignment was performed with the Burrows–Wheeler Aligner [13] (version 0.7.15-r1140). hs37d5 was used as the reference human genome. PCR duplicate reads were removed using Picard (version 2.9.0-1-gf5b9f50-SNAPSHOT, https://broadinstitute.github.io/picard/ (accessed on 1 February 2017)). Non-mappable reads were removed using SAMtools (version 1.3.1) [14]. After filtering out those reads, we applied the Genome Analysis Toolkit [15] (GATK version 3.5-0-g36282e4) base quality score recalibration and performed SNP and INDEL discovery (HaplotypeCaller). Finally, we identified 350, 346, and 343 variants in samples T1, N1, and Bl, respectively, and the variants were annotated using ANNOVAR (version 2016Feb1) [16]. As expected, the *KCNJ5* (p.G151R) mutation was identified in these three samples.

Variants that passed quality control were prioritized according to the following strategies. We only retained variants predicted to modify protein function; these included the nonsense, splice site, coding indel, and missense variants. We removed variants with minor allele frequencies >0.4% for the ESP6500 (ESP6500siv2\_all provided by ANNO-VAR) database, >0.4% for each population of the Exome Aggregation Consortium (exac03 provided by ANNOVAR), >0.4% in HGVD (containing genetic variations determined by exome sequencing of 1208 individuals in Japan) [17], and >0.4% in 2KJPN (whole-genome sequences of 2049 Japanese healthy individuals and construction of a highly accurate Japanese population reference panel). After removing these variants, we focused on variants identified only in the tumor sample. Variants that appeared to be mapping artifacts, and were too common in in-house controls, were also excluded from further analyses. Consequently, several somatic mutations were found in sample T1. Sanger sequencing of these genes confirmed mutations in catenin β 1 (*CTNNB1*), centromere protein E (*CENPE*), leucine zipper- and EF-hand-containing transmembrane protein 2 (*LETM2*), and ALG10 Alpha-1,2-Glucosyltransferase B (*ALG10B*) in T1 and T3, but not in T2, N1–N3, and Bl, suggesting that these genes might be associated with the rapid growth of the larger tumor. Among these genes, it is well known that mutation in *CTNNB1* is associated with tumor growth in adrenocortical carcinoma via the constitutively activated nuclear β catenin protein [18].

#### *3.3. Microarray Analyses*

Microarray analyses of T1–T3 (RNA#89–91, respectively) and N1–N3 (RNA#86 – 88) were performed using the Human Clariom™ S Array and GeneChip WT PLUS Reagent Kit (Thermo Fisher Scientific, #902916 and 902280) [7]. N1 was presumably contaminated with cells from the adrenal medulla, because a few genes known to be expressed in the adrenal medulla were highly expressed in N1 samples (e.g., tyrosine hydroxylase). Genes that exhibited a fold change of 1.3 or more in T1 and T3, as compared to N2 and N3, were used for pathway analysis using the Kyoto Encyclopedia of Genes and Genomes Database. Six pathways were significantly identified as upregulated, as follows: "Protein digestion and absorption" (*p* = 0.0016), "Renin-angiotensin system" (*p* = 0.0023), "Adipocytokine signaling pathway" (*p* = 0.0058), "Cell cycle" (*p* = 0.0091), "Pancreatic secretion" (*p* = 0.0171), and "p53 signaling pathway" (*p* = 0.0339). A similar analysis, using the WikiPathway Database, revealed three up-regulated pathways, as follows: "Retinoblastoma Gene in Cancer" (*p* = 0.0003), "Splicing factor NOVA regulated synaptic proteins" (*p* = 0.0091), and

"Deregulation of Rab and Rab Effector Genes in Bladder Cancer" (*p* = 0.0114). Upstream steroidogenic enzymes for aldosterone synthesis (i.e., cytochrome p450 family 11 subfamily A member 1 (CYP11A1), 3-β-hydroxysteroid dehydrogenase (HSD3B2), and 21-hydroxylase (CYP21A1)) did not exhibit variation in expression between samples, suggesting that the localization of aldosterone shown by MALDI-IMS (mainly in the subcapsular area but not in the tumors) was not due to a lack of the upstream steroidogenic enzyme expression, but other unidentified reasons. Overall, the status of gene variants and gene expression was consistent with the clinical course of the case.

#### *3.4. Confirmation of CYP11B2 Expression*

We compared the expression levels of *CYP11B2* mRNA between this case and archived APA cases, previously adrenalectomized in the Kansai Medical University (APA#7–APA#26), using qRT-PCR for *CYP11B2* (Supplementary Table S1). *CYP11B2*-expression levels in the tumors of cases APA#7, 18, 19, and 26 were lower than those in paired adjacent adrenal tissues, suggesting incorrect sampling or sampling from non-APA tumors; therefore, these samples were removed from the following analyses. Two tumors (tumors 1 and 2) were sampled from APA#9, but the *CYP11B2* expression of tumor 2 was lower than that of adjacent normal; therefore, tumor 2, but not tumor 1, was also removed from the following analyses. Normal adrenal APA#23 (APA#23N) showed the lowest *CYP11B2* expression level, and a fold difference of each sample (16 pairs) over APA#23N was calculated. Sanger sequencing of these cases for *KCNJ5* revealed that 10 cases harbored *KCNJ5* mutations (62.5%, p.G151R [*n* = 6], and p.L168R [*n* = 3], p.L168Hfs\*93 [*n* = 1]) in samples from tumors, but not in their paired adjacent adrenals. An average fold change of APA samples with *KCNJ5* mutation (513,214.9 ± 290,452.9 [mean ± S.D.]) was similar to that without *KCNJ5* mutation (708,321.1 ± 297,156.8, *p* = 0.319, unpaired Student's *t*-test using ∆∆Ct values). As expected from the results of CYP11B2 immunohistochemistry, *CYP11B2* expression levels were not different between non-tumor (816,286.5 ± 428,233.7-fold) and tumor portions (539,737.3 ± 336,381.8-fold) of the case (*p* = 0.393, unpaired Student's *t*-test using ∆∆Ct values). *CYP11B2* expression levels in the case (T1–T3 and N1–N3, 582,237 [interquartile range: 448,734–977,189]-fold) and APA (574,401 (328,933–817,145)-fold) were significantly higher than that of the paired adjacent normal adrenals (966 (62–11,986)-fold), and those in the case and APA were similar (Supplementary Figure S6). Consequently, whole enlarged adrenal in the case expressed high levels of CYP11B2 in mRNA and protein as APAs did.

#### **4. Result**

#### *4.1. Analyses of the Surgically Removed Adrenal Gland*

Comprehensive pathological and molecular analyses were approved by the institutional review boards. Immediately after surgery, the adrenal gland was cut into 16 pieces, as shown in Figure 1D,E. The adrenal gland had two apparent tumors (# and \* in Figure 1D,E) and many smaller nodules. Flash frozen tissues were also taken from three non-tumor portions (N1–N3 in Figure 1E), two portions from the larger tumor (T1 and T3), and one portion from the smaller tumor (T2). Frozen blocks, embedded in optimal cutting temperature compound, were prepared from four portions (FB5, FB10, FB15-1, and FB15-2 in Figure 1E), as previously reported [7,9]. The remaining adrenal tissues were fixed with 10% formalin (Supplementary Figure S1) and used for formalin-fixed paraffin-embedded (FFPE) blocks for regular pathological diagnosis. Sanger sequencing of *KCNJ5* was performed, as previously reported [11], and a de novo *KCNJ5* mutation (p.G151R) was detected in genomic DNA from N1–N3 (DNA #86–88, respectively), T1–T3 (#89–92, respectively), as well as her blood (Blood, #92) and hair root (#155), but not in blood samples from her mother (#93) and father (#156) (see "DNA and RNA isolation from flash frozen tissues, blood, and hair root" in the Materials and Methods, Supplementary Figure S2, and details of DNA samples shown in Supplementary Table S1). Histological analyses, using the frozen blocks (Figures 1F and S3) and FFPE tissues (Supplementary Figure S4), were performed. Microscopically, most of the tumor cells in the larger and smaller tumors (\* and
