*3.4. Familial Hyperaldosteronism Type IV*

Familial hyperaldosteronism type IV (FH type IV) is caused by gain-of-function mutations in Cav3.2, a T type Ca2+ channel encoded by *CACNA1H.* FH type IV was first identified in 2015 by exome sequencing of 40 unrelated subjects with early-onset hyperaldosteronism and hypertension (<10 years of age) [22]. Scholl et al. identified five subjects with the same heterozygous mutation in *CACNA1H* encoding the Ca2+ voltage gated channel (Cav3.2) resulting in a Cav3.2 p.Met1549Val substitution [16]. Comparisons of whole cell patch clamp recordings of Cav3.2 p.Met1549Val and wild-type Cav3.2 expressed in human embryonic kidney cells showed that the p.Met1549Val mutation causes an impairment of channel activation and inactivation. The mutant channel displayed slightly slower activation and much slower inactivation time constants compared with the wild-type channel as well as a tail current indicating that a proportion of the mutated channels remain non-inactivated. These properties would lead to an increase in Ca2+ influx in adrenal ZG cells and signal an increase in aldosterone production. Validation of this concept was subsequently demonstrated by the same group by expression of the Cav3.2 p.Met1549Val mutation in human adrenocortical (HAC15) cells which resulted in an increase in *CYP11B2* gene transcription and aldosterone secretion relative to cells expressing the wild-type channel [23]. Following the discovery by Scholl, additional mutations in *CACNA1H* were described involving a substitution of the same Met1549 residue (Met1549Ile) or other amino acid residues (Ser196Leu, Val1951Glu and Pro2083Leu) [24].

## **4. Somatic Mutations in Aldosterone-Producing Adenomas**

The most frequent genetic variation in APA is a somatic mutation of the *KCNJ5* gene [25]. First identified by Choi et al. in 2011 by exome sequencing, mutations in *KCNJ5* were identified in 8 of 22 APA resulting in GIRK p.Gly151Arg or p.Leu168Arg mutations [6]. Both mutations were demonstrated to interfere with the selectivity filter of the channel pore and result in membrane depolarization causing the opening of voltage gated Ca2+ channels in adrenal glomerulosa cells and Ca2+ influx [6]. Somatic *KCNJ5* mutations are found at a prevalence of 40–50% [26–30] although a higher prevalence has been reported in populations from Japan and China [31,32]. Following the description of the *KCNJ5* mutations, the application of next generation sequencing rapidly identified additional somatic mutations associated with aldosterone overproduction in sporadic PA. These include heterozygous gain-of-function mutations in Cav1.3 (the α1D subunit of the L-type voltage-dependent calcium channel) encoded by *CACNA1D* [8,9] and the ion transporters,

Na+/K+-ATPase (encoded by *ATP1A1*) and Ca2+-ATPase (encoded by *ATP2B3*) [7,8]. These mutations result in an increase in intracellular Ca2+ concentration thereby causing an increase in transcription of the *CYP11B2* gene that encodes aldosterone synthase. Activating mutations in exon 3 of *CTNNB1* that encodes β-catenin have been identified in APAs as well as in other adrenal tumours [33]. Despite these major advances, the mechanisms underlying the deregulated cell growth of APAs are probably not explained by somatic mutations and the GIRK4 Thr158Ala mutation does not enhance proliferation of adrenal cells in vitro [19]. Herein we discuss the transcriptome studies that have identified genes and signalling pathways that may function in the pathophysiology and pathogenesis of APA.

#### **5. Gene Expression Profiling**

Gene expression studies have identified genes with a potential role in the pathogenesis and pathophysiology of APAs (Table 2). Despite inter-study heterogeneity of gene expression data, which may be accounted for by the use of different reference tissues (adjacent cortex or normal adrenal tissue or, in some cases, non-functioning adrenocortical adenomas) and different diagnostic criteria [34–38], such studies have proven valuable in the identification of genes and signalling pathways with a potential role in the pathogenesis and pathophysiology of APAs.

Gene expression studies employing microarrays have shown a higher expression of *CYP11B2* in APA compared with normal adrenals [39–41] and by SAGE (serial analysis of gene expression) in an APA compared with adjacent cortical tissue [42]. However, another study of APA transcriptomes reported two distinct and opposing expression profiles for genes encoding steroidogenic enzymes with *CYP11B2* displaying increased or decreased expression levels with respect to normal adrenal tissue [43]. This apparent paradoxical decrease of the gene expressing aldosterone synthase in a tumour that overexpresses aldosterone may be accounted for by sampling areas of the normal adrenal reference tissue. In fact, these may contain aldosterone-producing cell clusters (APCC) that express high levels of *CYP11B2* with somatic *CACNA1D*, *ATP1A1* or *ATP2B3* mutations [44–46]. Conversely large APA with low expression of *CYP11B2* that give rise to inappropriate aldosterone production might occur. Another possibility is that non-APA nodules were used in the study due to non-selective diagnostic criteria.

Many studies have described an association of somatic APA mutations with histological phenotype. APAs carrying *KCNJ5* mutations have been widely reported to comprise predominantly large lipid-rich ZF-like cells (Figure 2) [8,47–50]. Some studies have also described a predominance of small compact ZG-like cells in APA harbouring *CACNA1D*, *ATP1A1* or *ATP2B3* mutations [8,27,49,51] and somatic APA genotype is associated with plasma steroid profiles [52]. Such genotype-phenotype associations indicate that APA genotype may influence transcriptome signatures. Histological differences between large lipid-rich ZF-like cells and small compact ZG-like cells in APA are shown in Figure 2.

**Figure 2.** Histopathological phenotype of aldosterone-producing adenomas. Haematoxylin and eosin staining of an aldosterone-producing adenoma showing large lipid-rich cells of the ZF type (indicated with arrows) (panel **A**) or a predominance of smaller compact cells of the ZG type (indicated with arrows) (panel **B**).

No differences in the transcriptome profiles of APA with and without *KCNJ5* mutations were initially described [26]. However, later studies reported distinct expression profiles of APA with *KCNJ5* mutations compared with APA without *KCNJ5* mutations (with higher *CYP11B2* expression in the tumours with *KCNJ5* mutations) [53]. Different expression profiles were reported in APA with *ATP1A1* and *ATP2B3* mutations relative to APA with *KCNJ5* mutations (with higher *CYP11B2* expression in the tumors with *ATP1A1* and *ATP2B3* mutations) [54]. Azizan et al. [35] demonstrated marked differences in *CYP17A1* gene expression from microarrays, validated by real-time PCR, in APA with a ZF phenotype compared to those APA with a ZG phenotype [47]. If *CYP17A1* and *CYP11B2* are expressed in the same cell then cortisol can be metabolized further to produce the hybrid steroids 18-hydroxycortisol and 18-oxocortisol [55]. Higher levels of these hybrid steroids are associated with FH type I and FH type III (although not in all cases) and in patients with an APA with a *KCNJ5* mutation [12,52].

*CYP17* expression in APA has been shown to be associated with APA phenotype with marked upregulation in adenomas comprising predominantly ZF-type cells [47]. *NURR1* (*NR4A2*, encoding Nur-related factor 1) and *NGFIB* (*NR4A1*, encoding nerve growth factor IB), genes that encode transcription factors playing a key role in the regulation of *CYP11B2* gene transcription [56], are upregulated in APA. Also, genes encoding the nuclear receptor transcription factors SF-1 (*NR5A1*) and DAX1 (*NR0B1*) that are essential for adrenal development and steroidogenesis, are upregulated in APA [39]. Although low DAX1 expression in adrenocortical tumours is associated with aldosterone production [57]. A target gene of SF-1, *VSNL1* [58], is upregulated in APA and *VSNL1* in vitro overexpression in the NCI H295R cell line results in an increase in aldosterone production under both basal and angiotensin II-stimulated conditions [59].

Several genes encoding G-protein-coupled receptors are among the genes upregulated in APA, including those encoding the luteinizing hormone receptor (LH-R encoded by *LHCGR*), gonadotropin releasing hormone receptor (GnRHR encoded by *GNRHR*), serotonin receptor 4 (HTR4), melanocortin 2 receptor (MC2R), and the angiotensin II type 1 receptor (AGTR1) [60]. Overexpression of LH-R in the adrenocortical carcinoma NCI H295R cell line causes a concentration-dependent increase in *CYP11B2* expression after stimulation with luteinizing hormone [40]. Accordingly, the expression of *LH-R* and *GnRHR* in APAs has been proposed to be related to increased aldosterone production during pregnancy [61]. Therefore, the presence of activating APA *CTNNB1* mutations might contribute to an abnormal receptor activation [60].

*NEFM*, encoding the medium neurofilament protein, is highly upregulated only in APAs without *KCNJ5* mutations and is selectively expressed in the ZG and in APA comprised of predominantly ZG cells [62,63]. Dopamine regulates aldosterone production via activation of its G-protein-coupled receptor (GPCR) subtypes and silencing of *NEFM* amplified aldosterone stimulation by a DR1 (dopamine receptor subunit 1) agonist and aldosterone secretion in response to the DR1 agonist was greater in primary cultures of APAs composed of primarily ZF cells compared with cultures of APAs with ZG cells. These data indicate a role for *NEFM* in aldosterone production and cell proliferation [63].

Analysis of the methylome of APAs demonstrated hypomethylation of GPCR genes and a strong association of promoter hypomethylation of the HTR4 and PTGER1 genes with the upregulation of mRNA levels, validated by real-time PCR, was demonstrated in APAs compared with non-functioning adrenocortical adenomas [64]. Methylation of HTR4 and PTGER1 was significantly inversely correlated with their respective mRNA expression levels [64]. The most hypomethylated promoter in APA is the *PCP4* (encoding purkinje cell protein 4) promoter with demethylation associated with enhanced gene transcription [65]. *CYP11B2* was also extensively hypomethylated in APAs [64] but although hypomethylation was not associated with gene expression levels in this study it could facilitate gene transcription [64]. In contrast, Howard et al., reported hypomethylation of APAs with hypomethylation and overexpression of *CYP11B2* [64].

Calcium is a key intracellular messenger for aldosterone production and the intracellular Ca2+ signaling pathway is independent of the renin–angiotensin–aldosterone system in APAs [66]. A number of genes involved in Ca2+ signaling or Ca2+ sequestration have been reported as upregulated in APAs and are described in more detail below. *VSNL1* that encodes a Ca2+-sensor protein and a target of the nuclear receptor SF-1 [58] was one of several upregulated genes in APAs by microarray analysis compared with normal adrenals validated by real-time PCR [41]. In NCI H295R adrenal cells, overexpression of *VSNL1* resulted in an upregulation of *CYP11B2* gene expression under both basal and angiotensin II-stimulated conditions thereby implicating a role for *VSNL1* in aldosterone production. Analysis of a larger sample set of tumours showed that *VSNL1* was overexpressed in APAs carrying a *KCNJ5* mutation compared with those APA without a *KCNJ5* mutation. A potential role for the calcium sensor in the protection of cells in an adenoma via Ca2+-related anti-apoptotic cell death mechanisms was hypothesized [59]. The expression of the VSNL1 protein in an APA (carrying a *KCNJ5* mutation) that displays strong CYP11B2 immunostaining is shown in Figure 3.

**Figure 3.** VSNL1 and CYP11B2 immunohistochemistry. An aldosterone-producing adenoma with a *KCNJ5* mutation stained with haematoxylin and eosin panels (**A**,**B**); immunostained for VSNL1, panels (**C**,**D**); and for CYP11B2 panels (**E**,**F**). The VSNL1 antibody was from Merck and the CYP11B2 was a kind gift from Prof Celso Gomez-Sanchez, University of Mississippi, Oxford, MS, USA.

The *CALN1* gene, that encodes the Ca2+ binding protein calneuron 1, has been reported as upregulated in APA in two transcriptome studies [41,67]. CALN1 was shown to potentiate aldosterone production and silencing *CALN1* led to a decrease in Ca2+ storage in the endoplasmic reticulum and abrogated angiotensin II-mediated aldosterone secretion in an adrenocortical carcinoma cell line [41].

*CALM2* encoding calmodulin 2 is a Ca2+-binding protein expressed in a wide-range of tissues involved in signalling, cell cycle progression and proliferation. *CALM2* was highly upregulated in a transcriptome comparison of APAs with the adjacent ZG [42]. The increased expression of *PCP4* in APA cells is likely to play a role in APA pathophysiology because PCP4 modulates Ca2+-binding by calmodulin and activates the calcium-calmodulin cascade leading to an increased expression of *CYP11B2* [68].

*GSTA1* (encoding glutathione-*S*-transferase, an enzyme that protects cells from reactive oxygen species, ROS) gene expression is inversely correlated with the level of aldosterone production in APAs with a *KCNJ5* mutation and appears to regulate aldosterone secretion via ROS and Ca2+ signalling [69]. *GSTA1* overexpression suppressed aldosterone biosynthesis, while silencing of *GSTA1* increased aldosterone production through increasing ROS, superoxide, H2O2 levels, Ca2+ influx and the expression of *CAMK1* (encoding Ca2+/calmodulin dependent protein kinase 1) and the transcription factors *NR4A1* (also called *NGFIB*) and *NR4A2* that regulate *CYP11B2* gene expression [69].

The epidermal growth factor-like teratocarcinoma-derived growth factor-1 gene (*TDGF1*) was identified as the most highly expressed gene in APAs compared with normal adrenals by microarray analysis [41]. *TDGF1* was also identified as upregulated in an APA relative to the paired adjacent cortex by serial analysis of gene expression [41]. Overexpression of *TDGF1* in NCI H295R adrenal cells activated the PI3K-Akt signalling pathway and led to an increase in aldosterone production, indicating a potential role in APA pathophysiology [41]. The activation of PI3K/Akt mTOR signalling, a pathway with a known role in cell proliferation, was also reported in patients with PA [41].

Wnt plays a key role in the development of the adrenal cortex and the dysregulation of this signalling pathway is associated with tumorigenesis [70]. The Wnt/β-catenin pathway is constitutively activated in around 70% of APAs [71] with the decreased expression on the Wnt inhibitor *SFRP2* (encoding secreted frizzled related protein 2) likely playing a role in the deregulated Wnt/β-catenin signalling [72]. *SFRP2* was also four-fold down-regulated in APAs compared with normal adrenals in an oligonucleotide microarray [72]. Mice with an ablation of *Sfrp2* display enhanced aldosterone production [72]. β-catenin appears to mediate aldosterone production by increasing the transcription of several genes including *AT1R*, *CYP21* and *CYP11B2* as well as upregulating expression of transcription factors NURR1 (*NR4A2*) and NUR77 (*NR4A1*) [72].

*NPNT* (nephronectin), a secreted matrix protein, was most highly expressed in APAs with a ZG phenotype with *CTNNB1* mutations. Thereby it may represent a potential biomarker to recognize a subtype of APAs and indicates a further mechanism by which the Wnt/β-catenin signalling pathway may upregulate aldosterone production [72]. These studies show that aberrant Wnt/β-catenin pathway activation is associated with APA development and suggests that the Wnt/β-catenin signalling mediates aldosterone production at multiple levels [71].


**Table 2.** Differentially expressed genes in aldosterone-producing adenomas and their reference tissues used in transcriptome studies.

AAC: adjacent adrenal cortex; APAs: aldosterone-producing adenomas; *KCNJ5*-mut APAs: APAs with *KCNJ5* mutations; NFA: non-functioning adrenocortical adenomas; NLA: normal adrenals; WT-*KCNJ5* APAs: APAs with wild type *KCNJ5* gene; ZF: *zona fasciculata*; ZG: *zona glomerulosa*.
