1. Primary aldosteronism
Aldosterone is synthesized in the adrenal cortex and plays an essential role in regulating blood pressure by promoting sodium reabsorption in the kidney. Primary aldosteronism (PA), which is a disorder of excess aldosterone secretion, is the most common form of secondary hypertension, with a prevalence of 5–10% among patients with hypertension
[1]. The risk of cardiometabolic and renal disease is higher in PA patients than in essential hypertension patients; thus, early diagnosis and appropriate treatment of PA are important for reducing its complications
[2][3][4][5]. PA is mainly classified into two subtypes: aldosterone-producing adenoma (APA) and bilateral idiopathic hyperaldosteronism (BHA). Although the etiology of PA has long remained unclear, recent developments in genetic analysis, including next-generation sequencing (NGS), have expanded our understanding of the genetic and molecular mechanisms of PA in the last decade. Exome sequencing discovered somatic mutations in
KCNJ5,
ATP1A1,
ATP2B3,
CACNA1D,
CACNA1H,
CLCN2, and
CTNNB1 in APA
[6][7][8][9][10][11][12][13]. Most of the causative genes encode ion channels or pumps, and their mutations lead to depolarization of the cell membrane due to impairment of ion transport. Depolarization activates voltage-gated Ca
2+ channels and intracellular calcium signaling and promotes the transcription of aldosterone synthase (
CYP11B2), resulting in overproduction of aldosterone (). Furthermore, some key molecules such as VSNL1, CALN1, GSTA1, NPNT, and CLGN have been detected in APA, and their functions in aldosterone production have been elucidated
[14][15][16][17][18]. Epigenetic regulation of
CYP11B2 has also been indicated in APA
[19][20][21][22].
Figure 1. Cellular mechanism of aldosterone synthesis in aldosterone-producing adenoma. Mutations of KCNJ5, ATP1A1, and CLCN2 lead to depolarization of the cell membrane due to impairment of ion transport. Depolarization activates voltage-gated Ca2+ channels and increases intracellular Ca2+ levels. Conversely, mutations of CACNA1D and CACNA1H directly cause an increase in Ca2+ conductance. ATP2B3 mutation reduces Ca2+ export from the cell. Activated calcium signaling promotes transcription of aldosterone synthase (CYP11B2), resulting in overproduction of aldosterone.
Familial hyperaldosteronism (FH) has also been reported as a rare cause of PA. There are four forms of FH (FH type 1 to type 4). Although it is rare, the study of FH was preferred as an approach to understand the pathophysiology of PA due to its heritability. The first report of FH was the case of a father and a son presenting the symptoms of PA in 1966, which was corrected by glucocorticoid treatment
[23]. Thus, this form of PA is called glucocorticoid-remediable aldosteronism (GRA) or FH type 1. In 1992, linkage analysis revealed that the molecular etiology of GRA was a chimeric gene composed of the promoter of 11β-hydroxylase (
CYP11B1) fused with the coding region of
CYP11B2, resulting in aldosterone overproduction regulated by ACTH
[24]. The chimeric
CYP11B1/CYP11B2 gene was not identified in APA
[25], whereas some causative genes, including
KCNJ5,
CLCN2, and
CACNA1H, have been discovered in the other forms of FH
[6][10][11][12].
2. KCNJ5
In 2011, Choi et al. analyzed 22 cases of APA using whole-exome sequencing and identified two recurrent somatic mutations of
KCNJ5 (G151R and L168R)
[6].
KCNJ5 encodes the G protein-coupled inwardly rectifying K
+ channel (GIRK4), which belongs to GIRK family members (GIRK1 to GIRK4). GIRK4, which consists of two membrane-spanning domains, one pore-forming region between the two transmembrane domains, and intracellular N and C termini, forms a channel as a homotetramer or heterotetramer with GIRK1. Both substitutions are located near the channel’s ion-selective filter and cause depolarization of the cell membrane due to the loss of ion selectivity of the K
+ channel and the increased intracellular influx of Na
+. The authors proposed that activated voltage-gated Ca
2+ channels resulting from these mutations promote autonomous secretion of aldosterone and cell proliferation. In subsequent studies with adrenocortical carcinoma cell lines, introduction of the
KCNJ5 mutation promoted aldosterone synthesis via depolarization of the cell membrane, allowing sodium and calcium influx into the cell
[26][27][28][29]. Mutated
KCNJ5 also increased the expression of
CYP11B2 with its transcription factors nuclear receptor related 1 (
Nurr1) and activating transcription factor 2 (
ATF2), and these stimulatory effects were inhibited by Ca
2+ channel blockers
[26][27][30]. Moreover, molecules related to calcium signaling, such as VSNL1 and CALN1, are highly expressed in APA, and they have important roles in aldosterone production
[14][15][31]. These results show that increased
CYP11B2 expression is mediated by the Ca
2+/calmodulin cascade. The relationship between
KCNJ5 mutation and cell proliferation is still controversial, and the difference in
KCNJ5 mutation modulation levels may influence adrenal cell growth
[26][32][33]. Several other
KCNJ5 mutations such as E145Q, I157del, and T158A have been reported, although G151R and L168R are the most frequent
[8][29][34][35][36][37][38][39][40][41][42][43][44][45].
KCNJ5 is the most commonly mutated somatic gene in Asians, Europeans, and Americans with APA
[38][41][45]. In a report of 474 APA cases from the European Network for the Study of Adrenal Tumors (ENS@T),
KCNJ5 mutation was found in 38% of cases
[45]. In White Americans and African Americans,
KCNJ5 mutation was found in 43% and 34% of cases, respectively
[37][42]. Conversely, reports from East Asia have shown that nearly 70% of APA patients have a
KCNJ5 mutation, with an ethnic difference
[41][43][46][47][48][49][50]. A meta-analysis showed that APA patients with
KCNJ5 mutation have phenotypic features of higher plasma aldosterone levels, young age, female sex, and larger tumor size
[51]. Subclinical hypercortisolism is sometimes accompanied by APA; aldosterone and cortisol co-producing adenoma has also been reported in
KCNJ5-mutated APA
[52]. However, a recent prospective study showed that subclinical hypercortisolism was common in APA without
KCNJ5 mutation or with a relatively larger tumor size
[53]. Cardiovascular complications in APA patients with
KCNJ5 mutations also have been evaluated in some studies. In
KCNJ5-mutated APA patients, higher left ventricular mass index (LVMI) and plasma aldosterone levels were reported than in those without
KCNJ5 mutation
[54]. Another group reported that the
KCNJ5-mutated group significantly improved LVMI after surgery
[55]. A recent study also showed that APA patients with
KCNJ5 mutations had higher LVMI and inappropriately excessive LVMI (ieLVMI), as well as a greater regression of LVMI and ieLVMI after adrenalectomy, in comparison to those without
KCNJ5 mutations in a propensity-score-matched cohort
[56]. These results indicate
KCNJ5 mutation is associated with left ventricular remodeling and diastolic function.
KCNJ5 mutation was also reported to be a predictor of hypertension remission after adrenalectomy for APA
[43][57]. On the other hand, subclinical hypercortisolism in patients with APA was indicated to be associated with a lower clinical complete success rate after adrenalectomy
[53].
The adrenal cortex comprises three morphologically and functionally distinct layers: zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR). Although the expressions of steroid enzymes are zone-specific, the histological features of APA are heterogeneous
[58]. CYP11B2 is specifically expressed in ZG, and 17α-hydroxylase/17,20-lyase (CYP17A1) is expressed in ZF and ZR in the normal adult adrenal gland; however, APA with a
KCNJ5 mutation typically has predominant clear cells (ZF-like cells)
[59], and expression of both CYP11B2 and CYP17A1 is found within the same tumor
[60][61]. Plasma levels of the hybrid steroids 18-oxocortisol and 18-hydroxycortisol have been reported to be higher in APA patients, particularly in
KCNJ5-mutated APA
[62], which could be explained by its ZF-significant phenotype (.)
[63]. Thus, steroids have been indicated as clinical biomarkers, and steroid profiling can be utilized for differentiating subtypes of PA
[64][65][66][67].
Figure 2. Scheme of steroidogenic pathways for aldosterone, 18-oxocortisol, and 18-hydroxycortisol. Both CYP11B2 (aldosterone synthase) and CYP17A1 (17α-hydroxylase/17,20-lyase) are required to synthesize 18-oxocortisol and 18-hydroxycortisol. Thus, plasma levels of 18-oxocortisol and 18-hydroxycortisol are likely to be higher in patients with KCNJ5-mutated aldosterone-producing adenoma (APA), while they are very low in normal adults. CYP11A1: cytochrome P450 cholesterol side-chain cleavage; CYP11B1: 11β-hydroxylase; CYP21A2: 21-hydroxylase; HSD3B2: 3β-hydroxysteroid dehydrogenase type 2; StAR: steroidogenic acute regulatory protein; ZF: zona fasciculata; ZG: zona glomerulosa.
Germline mutation in
KCNJ5 also has been identified in FH. In 2008, Geller et al. reported the case of a father and two daughters with a new form of PA
[68]. They showed early-onset PA and marked adrenocortical hyperplasia, which did not respond to medical therapy and led to bilateral adrenalectomy. Choi et al. genetically analyzed this family and discovered germline
KCNJ5 mutation responsible for the disease, which was later classified as FH type 3
[6]. Since then, various phenotypes of FH type 3 depending on genotype have been reported; T158A, I157S, E145Q, and G151R are reported to have severe early-onset PA with bilateral adrenal hyperplasia, requiring bilateral adrenalectomy
[6][69][70][71]. On the other hand, G151E and Y152C are associated with mild PA with no adrenal abnormalities on computed tomography (CT) scan and can be controlled by mineralocorticoid receptor antagonist (MRA)
[71][72][73]. In vitro study demonstrated that transduction of
KCNJ5 G151E leads to profoundly large Na
+ conductance compared with other mutations, leading to Na
+-influx-dependent cell lethality
[71][72]. Therefore, it is suggested that these marked alterations of channel function prevent the development of adrenal hyperplasia, resulting in a mild clinical phenotype. However, there was a report of the early-onset PA with de novo
KCNJ5 G151R germline mutation and no adrenal enlargement whose symptoms were successfully controlled by MRA, indicating that diverse clinical phenotype in FH type 3 cannot be defined solely by
KCNJ5 genotype
[74]. In addition, two cases of early-onset PA possibly caused by mosaicism for
KCNJ5 mutations were reported
[75][76].
3. ATP1A1
Beuschlein et al. identified a somatic mutation in
ATP1A1 in 16/308 (5.2%) APAs
[7], and Azizan et al. found it in 2 of 10 ZG-like APAs without
KCNJ5 mutation
[8]. In contrast to
KCNJ5-mutated APA, APA with
ATP1A1 mutation is more commonly found in males and has histological features of predominant ZG-like cells
[7][8].
ATP1A1 encodes the alpha 1 subunit of Na
+/K
+ ATPase, which transports three Na
+ ions in exchange for two K
+ ions. The alpha subunit is composed of 10 transmembrane domains (M1–M10) with intracellular N and C termini. Several somatic mutations such as G99R, L104R, V332G, and EETA963S were identified in the M1, M4, and M9 domains
[7][8][35]. Mutations in the M1 and M4 domains, which result in alteration of K
+ binding and loss of pump activity, lead to depolarization of the cell membrane and autonomous secretion of aldosterone
[7]. Mutations in the M9 domain affect a supposed Na
+-specific site, resulting in loss of pump activity
[8]. These mutations were suggested to lead to abnormal H
+ or Na
+ leakage current, which is a similar mechanism to that of the
KCNJ5 mutation
[8]. However, in vitro study using adrenocortical cells demonstrated that mutations in
ATP1A1 induce depolarization of the cell membrane and intracellular acidification due to H
+ leak, but not an overt increase in intracellular Ca
2+ [77]. The specific mechanism of this acidification in autonomous aldosterone production has not been clarified.
The frequency of
ATP1A1 mutation determined through Sanger sequencing performed on whole tumor sample DNA was not as high as that of
KCNJ5 reported previously. However, a recently developed sequencing method using targeted NGS performed on DNA extracted from formalin-fixed paraffin-embedded tissues expressing CYP11B2 in immunohistochemistry (IHC) has enabled the more frequent detection of somatic mutations in APA
[37]. The CYP11B2 IHC-guided targeted NGS approach identified 5.0–17% of
ATP1A1 mutations in APA cases
[37][42][78][79], whereas the frequency of
ATP1A1 mutations was 2.4–8.2% using conventional methods
[7][35][38][41][45]. There are few reports of specific clinical characteristics of APA patients with non-
KCNJ5 mutation; one report showed that APA patients with ATPase mutation tended to have more severe hyperaldosteronism compared to those with wild type, although the sample size was small
[80].
5. CACNA1D
Scholl et al. identified five somatic
CACNA1D mutations (G403R and I770M) among 43 APAs without
KCNJ5 mutation
[9].
CACNA1D encodes a calcium channel voltage-dependent L-type alpha-1D subunit, which contains four repeated domains (I–IV), each with six transmembrane segments (S1–S6). These altered residues locate in the S6 segments lining the channel pore and induce a shift in voltage-dependent gating to a more negative voltage, leading to an increase in intracellular Ca
2+ levels
[9]. However, subsequent studies have shown that somatic mutations in
CACNA1D are found throughout the gene in APA
[84]. Azizan et al. also reported somatic
CACNA1D mutations in ZG-like APA at the same time
[8]. They also reported that
CACNA1D mutations were associated with small tumor size, but this association was not found in a recent study using the CYP11B2 IHC-guided targeted NGS approach
[79]. The CYP11B2 IHC-guided targeted NGS approach identified a large number of
CACNA1D mutations (14–42%)
[37][42][78][79] compared to conventional methods (0.6–10.3%)
[38][41][45]. Moreover,
CACNA1D mutations are most prevalent (42%), followed by
KCNJ5 mutations, in African American patients with APA
[42].
Scholl et al. also reported de novo germline
CACNA1D mutations (G403D and I770M) in two children featuring early-onset PA with seizures and neurologic abnormalities (PASNA). Although several cases of neurodevelopmental disease with
CACNA1D de novo germline mutations have been reported, only four cases presenting early-onset PA have been described to date
[9][85][86]. Treatment with calcium channel blockers (amlodipine and nifedipine) normalized blood pressure in two of these cases
[9][86], and CT scan showed no adrenal abnormality in one case
[9].
6. CTNNB1
CTNNB1 encodes β-catenin, and its mutation induces constitutive activation of Wnt/β-catenin signaling. Although Wnt/β-catenin signaling plays a crucial role in normal development and maintenance of the adrenal cortex
[87], activated Wnt/β-catenin signaling is also observed in APA
[88][89]. In addition to ion channels and ATPases, mutations in
CTNNB1 have been reported in APA with 0–5.1% frequency
[13][37][42][78][79][90]. The extracellular matrix gene
NPNT, which is downstream of the Wnt/β-catenin signaling pathway, is upregulated in ZG-like APA, especially with
CTNNB1 mutation. NPNT over-expression increases aldosterone production in adrenal cells
[17].
CTNNB1 mutation has also been found in other adrenocortical adenomas and adrenocortical carcinomas
[91]. A previous study showed that transgenic mice with constitutive β-catenin activation in adrenal tumors develop hyperaldosteronism and malignancy
[92]. Taken together, these results suggest that
CTNNB1 mutations stimulate ZG cell proliferation and Wnt/β-catenin activation participates in aldosterone production. APA with
CTNNB1 mutation is more common in females and has variable histological features
[13][90]. A higher risk of residual hypertension after adrenalectomy in patients with
CTNNB1-mutated APA was shown in one report
[90]. Clinical and histological features of APA harboring each somatic mutation are summarized in .
Table 1. Clinical and histological features of APA harboring each somatic mutation.