13 Review Article Primary aldosteronism diagnostics: KCNJ5 mutations and hybrid steroid synthesis in aldosterone-producing adenomas Juilee Rege1, Adina F. Turcu2, William E. Rainey1,2 1Department of Molecular and Integrative Physiology, 2Division of Metabolism, Endocrinology, and Diabetes, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA Contributions: (I) Conception and design: J Rege, WE Rainey; (II) Administrative support: WE Rainey; (III) Provision of study materials or patients: None; (IV) Collection and assembly of data: J Rege, WE Rainey; (V) Data analysis and interpretation: None; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. Correspondence to: William E. Rainey, PhD. Departments of Molecular and Integrative Physiology and Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA. Email: [email protected]. Abstract: Primary aldosteronism (PA) is characterized by autonomous aldosterone production by renin- independent mechanisms and is most commonly sporadic. While 60–70% of sporadic PA can be attributed to bilateral hyperaldosteronism, the remaining 30–40% is caused by a unilateral aldosterone-producing adenoma (APA). Somatic mutations in or near the selectivity filter the KCNJ5 gene (encoding the potassium channel GIRK4) have been implicated in the pathogenesis of both sporadic and familial PA. Several studies using tumor tissue, peripheral and adrenal vein samples from PA patients have demonstrated that along with aldosterone, the hybrid steroids 18-hydroxycortisol (18OHF) and 18-oxocortisol (18oxoF) are a hallmark of APA harboring KCNJ5 mutations. Herein, we review the recent advances with respect to the molecular mechanisms underlying the pathogenesis of PA and the steroidogenic fingerprints of KCNJ5 mutations. In addition, we present an outlook toward the future of PA subtyping and diagnostic work-up utilizing steroid profiling. Keywords: Aldosterone-producing adenomas; KCNJ5; CYP11B2; CYP17A1; 18-hydroxycortisol; 18-oxocortisol; aldosterone Submitted Oct 22, 2019. Accepted for publication Nov 06, 2019. doi: 10.21037/gs.2019.10.22 View this article at: http://dx.doi.org/10.21037/gs.2019.10.22 Introduction carcinoma (13). Differentiation of the uni- and bilateral forms of PA is Primary aldosteronism (PA) is the most common form important for guiding therapy. Unilateral PA can benefit of secondary hypertension, and it accounts for 5–8% of from adrenalectomy and BHA requires indefinite medical hypertension (1-6) and 11–20% of resistant hypertension therapy, which typically incorporates a mineralocorticoid (7-9). PA is characterized by inappropriate autonomous receptor antagonists (MRA) (14,15). Adrenal vein sampling production of aldosterone via renin-independent (AVS) is the most reliable method for distinguishing between mechanisms (10). Most PA patients exhibit a sporadic APA and BHA (10). However, there are several caveats form, whereas 5–6% of the cases are caused by familial to the AVS methodology and interpretation of hormonal disease (11). Approximately 60–70% of PA cases can be data. Despite being highly predictive of outcome (10), this attributed to bilateral hyperaldosteronism (BHA), with the procedure is laborious, invasive, and expensive. Additionally, remaining 30–40% being caused by unilateral aldosterone- AVS is performed in a limited number of referral centers, and producing adenomas (APA) (2,12). Uncommon forms it is dependent on highly-skilled interventional radiologists of PA include unilateral adrenal hyperplasia and adrenal with large annual AVS volume (16-18). © Gland Surgery. All rights reserved. Gland Surg 2020;9(1):3-13 | http://dx.doi.org/10.21037/gs.2019.10.22 4 Rege et al. Steroid profiling of KCNJ5 mutations in aldosterone-producing adenomas Cholesterol StAR CYP11A1 CYP17A1 (17α-hydroxylase) Pregnenolone 17OH-Pregnenolone HSD3B2 CYP17A1 HSD3B2 (17α-hydroxylase) Progesterone 17OH-Progesterone CYP21A2 CYP21A2 11-Deoxycorticosterone 11-Deoxycortisol CYP11B2 CYP11B1/ (11β-hydroxylase) CYP11B2 (11β-hydroxylase) Corticosterone Cortisol CYP11B2 CYP11B1/ (18-hydroxylase) CYP11B2 (18-hydroxylase) 18OH-Corticosterone 18OH-Cortisol CYP11B2 (18-methyloxidase) CYP11B2 (18-methyloxidase) Aldosterone 18-oxocortisol Figure 1 The steroidogenic pathway for aldosterone and the ‘hybrid steroids’—18OH-cortisol and 18-oxocortisol. While only CYP11B2 is required for aldosterone production, both CYP17A1 and CYP11B2 contribute to the biosynthesis of the hybrid steroids. A simple blood test that identifies APA-derived serum in patients with familial hyperaldosteronism type 1 steroid biomarkers would help distinguish patients who (FH type I) (31,32). FH type I accounts for <1% of will benefit from adrenalectomy from those who should be cases of PA (11) and is caused by a chimeric gene that is treated with medical therapy. Such biomarkers could also composed of the promoter of 11β-hydroxylase (CYP11B1) conserve healthcare resources by sparing many patients fused with the coding region of aldosterone synthase expensive imaging and invasive studies. Finally, the utility (CYP11B2) (31). Like CYP11B1, this CYP11B1/CYP11B2 of such biomarkers would increase the rate of PA screening, chimeric enzyme is present in the zona fasciculata (ZF) of facilitate PA diagnosis and appropriate treatment, and thus the adrenal cortex and is regulated by ACTH. As a result, reduce the burden of cardiovascular and renal complications the CYP11B1/CYP11B2 chimera is able to use cortisol as a which affect PA patents disproportionately more than those substrate to produce 18OHF, which is further metabolized with essential hypertension. to 18oxoF (19,23,30,32-34) (Figure 1). These metabolites of cortisol are designated as “hybrid” steroids owing to their molecular structure comprising features of steroid Hybrid steroids as diagnostic markers for metabolism which typically occur in the zona glomerulosa differentiation of APA from BHA (ZG) (18-hydroxylation and 18-oxidation) and the ZF Over the past 25 years, the applicability of the (17-hydroxylation) (31) (Figure 1). 18-oxygenated derivatives of cortisol, 18-oxocortisol In the normal adrenal gland, expression of CYP11B2 is (18oxoF) and 18-hydroxycortisol (18OHF) for subtyping restricted to the ZG, while 17α-hydroxylase/17,20-lyase PA as uni- or bilateral, has been of interest (19-27). Several (CYP17A1) and CYP11B1 are expressed exclusively in the studies have shown higher levels of 18OHF and 18oxoF in ZF and zona reticularis (Figure 2), thereby leading to low PA patients compared to those with essential hypertension production of 18OHF and 18oxoF in normal subjects. (23,28-30). High concentrations were also demonstrated Histologic studies have shown that some APA display a ZG- © Gland Surgery. All rights reserved. Gland Surg 2020;9(1):3-13 | http://dx.doi.org/10.21037/gs.2019.10.22 Gland Surgery, Vol 9, No 1 February 2020 5 HE CYP11B2 CYP17A1 A B C Normal Adrenal D E F APA mKCNJ5 Figure 2 Histologic findings in a normal adrenal (A,B,C) and an APA harboring a KCNJ5 mutation (D,E,F). Immunohistochemical analysis illustrates that CYP11B2 expression is localized in the zona glomerulosa of the normal adrenal (B), while CYP17A1 is expressed in the zona fasciculata and zona reticularis (C). A KCNJ5 mutation-harboring APA showed intratumoral CYP11B2 expression heterogeneity, with distinct CYP11B2 positive and negative regions within the tumor (E). The APA also exhibits elevated CYP17A1 expression in the tumor region. Scale: 5 mm. like phenotype, with small, compact cells, while other APA utility of these steroids as discriminators between the two PA are composed of large, lipid-rich cells, similar to those seen subtypes (24). Nevertheless, this analysis presented a composite in ZF (35-38). Although transcriptomic analysis has not of 12 steroids that was able to correctly classify the PA subtype detected any differences in CYP11B2 mRNA expression in 80% of the patients (24). A subsequent study from the same between the different APA subtypes (39), higher CYP11B2 group revealed that APA with different underlying somatic protein expression has been observed in the APA with ZG- mutations produce specific steroid fingerprints (44). like cells in some studies (35,37). Specific to APA with ZF-like histology is a higher expression of steroidogenic The hybrid steroids and KCNJ5 somatic enzymes required for cortisol biosynthesis, such as mutation connection in APA CYP17A1 and CYP11B1 (40-42). The co-expression of CYP17A1 and CYP11B2 in these APA facilitates the In 2016, Williams et al. identified specific steroid production of hybrid steroids (21,33,43) (Figures 1,2). fingerprints in adrenal vein (AV) and peripheral vein (PV) Ulick et al. performed the initial PA hybrid steroid plasma from patients with APA with various underlying studies in 1993 and demonstrated that urinary 18OHF and mutations (44). Of the 79 PA patients with unilateral PA 18oxoF were elevated in patients with APA compared to included, 34% had APA harboring KCNJ5 (encoding those with BHA (21,22). Numerous immunoassay studies the G protein-coupled inward-rectifying potassium followed that indicated higher plasma and urinary 18OHF channel 4, GIRK4) mutations, 11% had ATPase (ATP1A1 and 18oxoF levels in subjects with APA vs. those with BHA (Na+/K+ ATPase α1-subunit), ATP2B3 (Ca2+ ATPase 3) (19,23,29). More recently liquid chromatography-tandem mutations and 9% had CACNA1D (encoding the voltage- mass spectrometry (LC-MS/MS) was used to quantify dependent L-type calcium