Adrenal Anion Channels: New Roles in Zona Glomerulosa Physiology and in the Pathophysiology of Primary Aldosteronism.

The mineralocorticoid aldosterone is produced in the zona glomerulosa of the adrenal cortex. Its synthesis is regulated by the serum concentrations of the peptide hormone angiotensin II and potassium. The primary role of aldosterone is to control blood volume and electrolytes. The autonomous production of aldosterone (primary aldosteronism, PA) is considered the most frequent cause of secondary hypertension. Aldosterone-producing adenomas and (micro-)nodules are frequent causes of PA and often carry somatic mutations in ion channels and transporters. Rare familial forms of PA are due to germline mutations. Both somatic and germline mutations in the chloride channel gene CLCN2, encoding ClC-2, have been identified in PA. Clinical findings and results from cell culture and animal models have advanced our knowledge about the role of anions in PA. The zona glomerulosa of the adrenal gland has now been firmly established as a tissue in which anions play a significant role for signaling. In this overview, we aim to summarize the current knowledge and highlight novel concepts as well as open questions.

Isradipine therapy in Cacna1dIle772Met/+ mice ameliorates primary aldosteronism and neurologic abnormalities.

Somatic gain-of-function mutations in the L-type calcium channel CaV1.3 (CACNA1D gene) cause adrenal aldosterone-producing adenomas and micronodules. De novo germline mutations are found in a syndrome of primary aldosteronism, seizures, and neurologic abnormalities (PASNA) as well as in autism spectrum disorder. Using CRISPR/Cas9, we here generated mice with a Cacna1d gain-of-function mutation found in both adenomas and PASNA syndrome (Cacna1dIle772Met/+). These mice show reduced body weight and increased mortality from weaning to approximately 100 days of age. Male mice do not breed, likely due to neuromotor impairment, and the offspring of female mice die perinatally, likely due to lack of maternal care. Mice generated by in vitro fertilization showed elevated intracellular calcium in the aldosterone-producing zona glomerulosa, an elevated aldosterone/renin ratio, and persistently elevated serum aldosterone on a high-salt diet as signs of primary aldosteronism. Anesthesia with ketamine and xylazine induced tonic-clonic seizures. Neurologic abnormalities included hyperlocomotion, impaired performance in the rotarod test, impaired nest building, and slight changes in social behavior. Intracellular calcium in the zona glomerulosa, aldosterone levels, and rotarod performance responded to treatment with the calcium channel blocker isradipine, with implications for the therapy of patients with aldosterone-producing lesions and with PASNA syndrome.

CaV3.2 (CACNA1H) in Primary Aldosteronism.

Aldosterone is a steroid hormone produced in the zona glomerulosa (ZG) of the adrenal cortex. The most prominent function of aldosterone is the control of electrolyte homeostasis and blood pressure via the kidneys. The primary factors regulating aldosterone synthesis are the serum concentrations of angiotensin II and potassium. The T-type voltage-gated calcium channel CaV3.2 (encoded by CACNA1H) is an important component of electrical as well as intracellular calcium oscillations, which govern aldosterone production in the ZG. Excessive aldosterone production that is (partially) uncoupled from physiological stimuli leads to primary aldosteronism, the most common cause of secondary hypertension. Germline gain-of-function mutations in CACNA1H were identified in familial hyperaldosteronism, whereas somatic mutations are a rare cause of aldosterone-producing adenomas. In this review, we summarize these findings, put them in perspective, and highlight missing knowledge.

Genetics of Primary Aldosteronism.

Primary aldosteronism is considered the commonest cause of secondary hypertension. In affected individuals, aldosterone is produced in an at least partially autonomous fashion in adrenal lesions (adenomas, [micro]nodules or diffuse hyperplasia). Over the past decade, next-generation sequencing studies have led to the insight that primary aldosteronism is largely a genetic disorder. Sporadic cases are due to somatic mutations, mostly in ion channels and pumps, and rare cases of familial hyperaldosteronism are caused by germline mutations in an overlapping set of genes. More than 90% of aldosterone-producing adenomas carry somatic mutations in K+ channel Kir3.4 (KCNJ5), Ca2+ channel CaV1.3 (CACNA1D), alpha-1 subunit of the Na+/K+ ATPase (ATP1A1), plasma membrane Ca2+ transporting ATPase 3 (ATP2B3), Ca2+ channel CaV3.2 (CACNA1H), Cl- channel ClC-2 (CLCN2), ß-catenin (CTNNB1), and/or G-protein subunits alpha q/11 (GNAQ/11). Mutations in some of these genes have also been identified in aldosterone-producing (micro)nodules, suggesting a disease continuum from a single cell, acquiring a somatic mutation, via a nodule to adenoma formation, and from a healthy state to subclinical to overt primary aldosteronism. Individual glands can have multiple such lesions, and they can occur on both glands in bilateral disease. Familial hyperaldosteronism, typically with early onset, is caused by germline mutations in steroid 11-beta hydroxylase/ aldosterone synthase (CYP11B1/2), CLCN2, KCNJ5, CACNA1H, and CACNA1D.

Diagnosis and treatment of primary aldosteronism.

Primary aldosteronism is a common cause of secondary hypertension associated with excess cardiovascular morbidities. Primary aldosteronism is underdiagnosed because it does not have a specific, easily identifiable feature and clinicians can be poorly aware of the disease. The diagnostic investigation is a multistep process of screening, confirmatory testing, and subtype differentiation of unilateral from bilateral forms for therapeutic management. Adrenal venous sampling is key for reliable subtype identification, but can be bypassed in patients with specific characteristics. For unilateral disease, surgery offers the possibility of cure, with total laparoscopic unilateral adrenalectomy being the treatment of choice. Bilateral forms are treated mainly with mineralocorticoid receptor antagonists. The goals of treatment are to normalise both blood pressure and excessive aldosterone production, and the primary aims are to reduce associated comorbidities, improve quality of life, and reduce mortality. Prompt diagnosis of primary aldosteronism and the use of targeted treatment strategies mitigate aldosterone-specific target organ damage and with appropriate patient management outcomes can be excellent. Advances in molecular histopathology challenge the traditional concept of primary aldosteronism as a binary disease, caused by either a unilateral aldosterone-producing adenoma or bilateral adrenal hyperplasia. Somatic mutations drive autonomous aldosterone production in most adenomas. Many of these same mutations have been identified in nodular lesions adjacent to an aldosterone-producing adenoma and in patients with bilateral disease. In addition, germline mutations cause rare familial forms of aldosteronism (familial hyperaldosteronism types 1-4). Genetic testing for inherited forms in suspected cases of familial hyperaldosteronism avoids the burdensome diagnostic investigation in positive patients. In this Review, we discuss advances and future management approaches in the diagnosis of primary aldosteronism.

Subcellular localization of fibroblast growth factor receptor type 2 and correlation with CTNNB1 genotype in adrenocortical carcinoma.

OBJECTIVE: Fibroblast growth factor receptor (FGFR) 2 regulates the development of the adrenal gland in mice. In addition, FGFR2-mediated signalling has been shown to prevent apoptosis and to enhance proliferation in adrenocortical precursor cells. The activation of the Wingless/Int-1 (WNT)/beta catenin pathway as a key mechanism of adrenocortical tumourigenesis has been linked to FGFR2 signalling in other cell types. Therefore we hypothesised that FGFR2 expression may also play a role in adrenocortical carcinoma (ACC). We conducted a pilot study and analysed protein expression of FGFR2 in 26 ACCs using immunohistochemistry technique. Data on the CTNNB1 mutation status and clinical data were correlated to the expression of FGFR2. RESULTS: We observed a high variability in FGFR2 expression between the different tumour samples. There was a subset of ACC with comparatively high nuclear expression of FGFR2. We did not find a clear association between the CTNNB1 mutational status or clinical features and the FGFR2 expression. We conclude that FGFR signalling plays a role in adrenocortical carcinoma. Our data encourages further investigations of FGFR signalling in ACC, especially since new inhibitors of FGFR signalling are already entering clinical trials for the treatment of other cancer types.

Generation and characterization of a mitotane-resistant adrenocortical cell line.

Mitotane is the only drug approved for the therapy of adrenocortical carcinoma (ACC). Its clinical use is limited by the occurrence of relapse during therapy. To investigate the underlying mechanisms in vitro, we here generated mitotane-resistant cell lines. After long-term pulsed treatment of HAC-15 human adrenocortical carcinoma cells with 70 µM mitotane, we isolated monoclonal cell populations of treated cells and controls and assessed their respective mitotane sensitivities by MTT assay. We performed exome sequencing and electron microscopy, conducted gene expression microarray analysis and determined intracellular lipid concentrations in the presence and absence of mitotane. Clonal cell lines established after pulsed treatment were resistant to mitotane (IC50 of 102.2 ± 7.3 µM (n = 12) vs 39.4 ± 6.2 µM (n = 6) in controls (biological replicates, mean ± s.d., P = 0.0001)). Unlike nonresistant clones, resistant clones maintained normal mitochondrial and nucleolar morphology during mitotane treatment. Resistant clones largely shared structural and single nucleotide variants, suggesting a common cell of origin. Resistance depended, in part, on extracellular lipoproteins and was associated with alterations in intracellular lipid homeostasis, including levels of free cholesterol, as well as decreased steroid production. By gene expression analysis, resistant cells showed profound alterations in pathways including steroid metabolism and transport, apoptosis, cell growth and Wnt signaling. These studies establish an in vitro model of mitotane resistance in ACC and point to underlying molecular mechanisms. They may enable future studies to overcome resistance in vitro and improve ACC treatment in vivo.

Genetic causes of primary aldosteronism.

Primary aldosteronism is characterized by at least partially autonomous production of the adrenal steroid hormone aldosterone and is the most common cause of secondary hypertension. The most frequent subforms are idiopathic hyperaldosteronism and aldosterone-producing adenoma. Rare causes include unilateral hyperplasia, adrenocortical carcinoma and Mendelian forms (familial hyperaldosteronism). Studies conducted in the last eight years have identified somatic driver mutations in a substantial portion of aldosterone-producing adenomas, including the genes KCNJ5 (encoding inwardly rectifying potassium channel GIRK4), CACNA1D (encoding a subunit of L-type voltage-gated calcium channel CaV1.3), ATP1A1 (encoding a subunit of Na+/K+-ATPase), ATP2B3 (encoding a Ca2+-ATPase), and CTNNB1 (encoding ß-catenin). In addition, aldosterone-producing cells were recently reported to form small clusters (aldosterone-producing cell clusters) beneath the adrenal capsule. Such clusters accumulate with age and appear to be more frequent in individuals with idiopathic hyperaldosteronism. The fact that they are associated with somatic mutations implicated in aldosterone-producing adenomas also suggests a precursor function for adenomas. Rare germline variants of CYP11B2 (encoding aldosterone synthase), CLCN2 (encoding voltage-gated chloride channel ClC-2), KCNJ5, CACNA1H (encoding a subunit of T-type voltage-gated calcium channel CaV3.2), and CACNA1D have been reported in different subtypes of familial hyperaldosteronism. Collectively, these studies suggest that primary aldosteronism is largely due to genetic mutations in single genes, with potential implications for diagnosis and therapy.

CLCN2 clicks with aldosterone-producing adenomas, too!

Germline mutations in the chloride channel gene CLCN2 have been described as cause of familial hyperaldosteronism type II. In this issue, Dutta and colleagues in a groundbreaking study identify a somatic (tumor-specific) CLCN2 mutation in an aldosterone-producing adenoma, expanding the disease spectrum associated with CLCN2 mutations.

USP8 Mutations in Pituitary Cushing Adenomas-Targeted Analysis by Next-Generation Sequencing.

Gain-of-function somatic mutations in the ubiquitin specific protease 8 (USP8) gene have recently been reported as a cause of pituitary adenomas in Cushing disease. Molecular diagnostic testing of tumor tissue may aid in the diagnosis of specimens obtained through therapeutic transsphenoidal surgery; however, for small tumors, availability of fresh tissue is limited, and contamination with normal tissue is frequent. We performed molecular testing of DNA isolated from single formalin-fixed and paraffin-embedded (FFPE) tissue sections of 42 pituitary adenomas from patients with Cushing disease (27 female patients and 15 male patients; mean age at surgery, 42.5 years; mean tumor size, 12.2 mm). By Sanger sequencing, we identified previously reported USP8 missense mutations in six tumors. Targeted next-generation sequencing (NGS) revealed known or previously undescribed missense mutations in three additional tumors (two with two different mutations each), with mutant allele frequencies as low as 3%. Of the nine tumors with USP8 mutations (mutation frequency, 21.4%), seven were from female patients (mutation frequency, 25.9%), and two were from male patients (mutation frequency, 13.3%). Mutant tumors were on average 11.4 mm in size, and patients with mutations were on average 43.9 years of age. The overall USP8 mutation frequency in our cohort was lower than in previously described cohorts, and we did not observe USP8 deletions that were frequent in other cohorts. We demonstrate that testing for USP8 variants can be performed from small amounts of FFPE tissue. NGS showed higher sensitivity for USP8 mutation detection than did Sanger sequencing. Assessment for USP8 mutations may complement histopathological diagnosis.

Unanswered Questions in the Genetic Basis of Primary Aldosteronism.

Over the past six years, the genetic basis of a significant fraction of primary aldosteronism (PA) cases has been solved. Breakthrough discoveries include the role of somatic variants in the KCNJ5, CACNA1D, ATP1A1, and ATP2B3 genes as causes of aldosterone-producing adenomas (APAs), and the recognition of three novel hyperaldosteronism syndromes with germline variants in the KCNJ5, CACNA1D, and CACNA1H genes. The description of somatic variants in CACNA1D and ATP1A1 in aldosterone-producing cell clusters (APCCs) suggests that these clusters are precursors of some aldosterone-producing adenomas. Yet, a number of questions remain unanswered. These include the genetic basis of about 40% of APAs without somatic variants in known genes. Do technical issues explain this finding, or are the unexplained APAs due to somatic copy number variation or rare variants in thus-far undiscovered genes? Similarly, the role of CTNNB1 (beta catenin) variants in APA pathogenesis is still unclear. The major question to be solved is the genetic basis of bilateral adrenal hyperplasia (BAH). Is BAH due to the bilateral occurrence of APCCs, to germline variants, or perhaps due to unknown serum factors? Lastly, the etiology of unsolved cases of apparently familial hyperaldosteronism remains to be discovered. It is expected that genetic studies over the next few years will lead to answers to at least some of the questions raised.

Macrolides selectively inhibit mutant KCNJ5 potassium channels that cause aldosterone-producing adenoma.

Aldosterone-producing adenomas (APAs) are benign tumors of the adrenal gland that constitutively produce the salt-retaining steroid hormone aldosterone and cause millions of cases of severe hypertension worldwide. Either of 2 somatic mutations in the potassium channel KCNJ5 (G151R and L168R, hereafter referred to as KCNJ5MUT) in adrenocortical cells account for half of APAs worldwide. These mutations alter channel selectivity to allow abnormal Na+ conductance, resulting in membrane depolarization, calcium influx, aldosterone production, and cell proliferation. Because APA diagnosis requires a difficult invasive procedure, patients often remain undiagnosed and inadequately treated. Inhibitors of KCNJ5MUT could allow noninvasive diagnosis and therapy of APAs carrying KCNJ5 mutations. Here, we developed a high-throughput screen for rescue of KCNJ5MUT-induced lethality and identified a series of macrolide antibiotics, including roxithromycin, that potently inhibit KCNJ5MUT, but not KCNJ5WT. Electrophysiology demonstrated direct KCNJ5MUT inhibition. In human aldosterone-producing adrenocortical cancer cell lines, roxithromycin inhibited KCNJ5MUT-induced induction of CYP11B2 (encoding aldosterone synthase) expression and aldosterone production. Further exploration of macrolides showed that KCNJ5MUT was similarly selectively inhibited by idremcinal, a macrolide motilin receptor agonist, and by synthesized macrolide derivatives lacking antibiotic or motilide activity. Macrolide-derived selective KCNJ5MUT inhibitors thus have the potential to advance the diagnosis and treatment of APAs harboring KCNJ5MUT.

A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis.

BACKGROUND: Dysregulated WNT signaling dominates adrenocortical malignancies. This study investigates whether silencing of the WNT negative regulator DKK3 (Dickkopf-related protein 3), an implicated adrenocortical differentiation marker and an established tumor suppressor in multiple cancers, allows dedifferentiation of the adrenal cortex. METHODS: We analyzed the expression and regulation of DKK3 in human adrenocortical carcinoma (ACC) by qRT-PCR, immunofluorescence, promoter methylation assay, and copy number analysis. We also conducted functional studies on ACC cell lines, NCI-H295R and SW-13, using siRNAs and enforced DKK3 expression to test DKK3's role in blocking dedifferentiation of adrenal cortex. RESULTS: While robust expression was observed in normal adrenal cortex, DKK3 was down-regulated in the majority (>75%) of adrenocortical carcinomas (ACC) tested. Both genetic (gene copy loss) and epigenetic (promoter methylation) events were found to play significant roles in DKK3 down-regulation in ACCs. While NCI-H295R cells harboring ß-catenin activating mutations failed to respond to DKK3 silencing, SW-13 cells showed increased motility and reduced clonal growth. Conversely, exogenously added DKK3 also increased motility of SW-13 cells without influencing their growth. Enforced over-expression of DKK3 in SW-13 cells resulted in slower cell growth by an extension of G1 phase, promoted survival of microcolonies, and resulted in significant impairment of migratory and invasive behaviors, largely attributable to modified cell adhesions and adhesion kinetics. DKK3-over-expressing cells also showed increased expression of Forkhead Box Protein O1 (FOXO1) transcription factor, RNAi silencing of which partially restored the migratory proficiency of cells without interfering with their viability. CONCLUSIONS: DKK3 suppression observed in ACCs and the effects of manipulation of DKK3 expression in ACC cell lines suggest a FOXO1-mediated differentiation-promoting role for DKK3 in the adrenal cortex, silencing of which may allow adrenocortical dedifferentiation and malignancy.

Intracellular Molecular Differences in Aldosterone- Compared to Cortisol-Secreting Adrenal Cortical Adenomas.

The adrenal cortex is a major site of steroid hormone production. Two hormones are of particular importance: aldosterone, which is produced in the zona glomerulosa in response to volume depletion and hyperkalemia, and cortisol, which is produced in the zona fasciculata in response to stress. In both cases, acute stimulation leads to increased hormone production, and chronic stimulation causes hyperplasia of the respective zone. Aldosterone- and cortisol-producing adenomas (APAs and CPAs) are benign tumors of the adrenal cortex that cause excess hormone production, leading to primary aldosteronism and Cushing's syndrome, respectively. About 40% of the APAs carry somatic heterozygous gain-of-function mutations in the K(+) channel KCNJ5. These mutations lead to sodium permeability, depolarization, activation of voltage-gated Ca(2+) channels, and Ca(2+) influx. Mutations in the Na(+)/K(+)-ATPase subunit ATP1A1 and the plasma membrane Ca(2+)-ATPase ATP2B3 similarly cause Na(+) or H(+) permeability and depolarization, whereas mutations in the Ca(2+) channel CACNA1D directly lead to increased calcium influx. One in three CPAs carries a recurrent gain-of-function mutation (L206R) in the PRKACA gene, encoding the catalytic subunit of PKA. This mutation causes constitutive PKA activity by abolishing the binding of the inhibitory regulatory subunit to the catalytic subunit. These mutations activate pathways that are relatively specific to the respective cell type (glomerulosa versus fasciculata), and there is little overlap in mutation spectrum between APAs and CPAs, but co-secretion of both hormones can occur. Mutations in CTNNB1 (beta-catenin) and GNAS (Gsα) are exceptions, as they can cause both APAs and CPAs through pathways that are incompletely understood.

CACNA1H(M1549V) Mutant Calcium Channel Causes Autonomous Aldosterone Production in HAC15 Cells and Is Inhibited by Mibefradil.

We recently demonstrated that a recurrent gain-of-function mutation in a T-type calcium channel, CACNA1H(M1549V), causes a novel Mendelian disorder featuring early-onset primary aldosteronism and hypertension. This variant was found independently in five families. CACNA1H(M1549V) leads to impaired channel inactivation and activation at more hyperpolarized potentials, inferred to cause increased calcium entry. We here aimed to study the effect of this variant on aldosterone production. We heterologously expressed empty vector, CACNA1H(WT) and CACNA1H(M1549V) in the aldosterone-producing adrenocortical cancer cell line H295R and its subclone HAC15. Transfection rates, expression levels, and subcellular distribution of the channel were similar between CACNA1H(WT) and CACNA1H(M1549V). We measured aldosterone production by an ELISA and CYP11B2 (aldosterone synthase) expression by real-time PCR. In unstimulated cells, transfection of CACNA1H(WT) led to a 2-fold increase in aldosterone levels compared with vector-transfected cells. Expression of CACNA1H(M1549V) caused a 7-fold increase in aldosterone levels. Treatment with angiotensin II or increased extracellular potassium levels further stimulated aldosterone production in both CACNA1H(WT)- and CACNA1H(M1549V)-transfected cells. Similar results were obtained for CYP11B2 expression. Inhibition of CACNA1H channels with the T-type calcium channel blocker Mibefradil completely abrogated the effects of CACNA1H(WT) and CACNA1H(M1549V) on CYP11B2 expression. These results directly link CACNA1H(M1549V) to increased aldosterone production. They suggest that calcium channel blockers may be beneficial in the treatment of a subset of patients with primary aldosteronism. Such blockers could target CACNA1H or both CACNA1H and the L-type calcium channel CACNA1D that is also expressed in the adrenal gland and mutated in patients with primary aldosteronism.

Novel somatic mutations in primary hyperaldosteronism are related to the clinical, radiological and pathological phenotype.

UNLABELLED: Aldosterone-producing adenomas (APAs) and bilateral adrenal hyperplasia are important causes of secondary hypertension. Somatic mutations in KCNJ5, CACNA1D, ATP1A1, ATP2B3 and CTNNB1 have been described in APAs. OBJECTIVE: To characterize clinical-pathological features in APAs and unilateral adrenal hyperplasia, and correlate them with genotypes. DESIGN: Retrospective study. SUBJECTS AND MEASUREMENTS: Clinical and pathological characteristics of 90 APAs and seven diffusely or focally hyperplastic adrenal glands were reviewed, and samples were examined for mutations in known disease genes by Sanger or exome sequencing. RESULTS: Mutation frequencies were as follows: KCNJ5, 37·1%; CACNA1D, 10·3%; ATP1A1, 8·2%; ATP2B3, 3·1%; and CTNNB1, 2·1%. Previously unidentified mutations included I157K, F154C and two insertions (I150_G151insM and I144_E145insAI) in KCNJ5, all close to the selectivity filter, V426G_V427Q_A428_L433del in ATP2B3 and A39Efs*3 in CTNNB1. Mutations in KCNJ5 were associated with female and other mutations with male gender (P = 0·007). On computed tomography, KCNJ5-mutant tumours displayed significantly greater diameter (P = 0·023), calculated area (P = 0·002) and lower precontrast Hounsfield units (P = 0·0002) vs tumours with mutations in other genes. Accordingly, KCNJ5-mutant tumours were predominantly comprised of lipid-rich fasciculata-like clear cells, whereas other tumours were heterogeneous (P = 5 × 10(-6) vs non-KCNJ5 mutant and P = 0·0003 vs wild-type tumours, respectively). CACNA1D mutations were present in two samples with hyperplasia without adenoma. CONCLUSIONS: KCNJ5-mutant tumours appear to be associated with fasciculata-like clear cell predominant histology and tend to be larger with a characteristic imaging phenotype. Novel somatic KCNJ5 variants likely cause adenomas by loss of potassium selectivity, similar to previously described mutations.

PRKACA mutations in cortisol-producing adenomas and adrenal hyperplasia: a single-center study of 60 cases.

OBJECTIVE: Cortisol excess due to adrenal adenomas or hyperplasia causes Cushing's syndrome. Recent genetic studies have identified a somatic PRKACA(L206R) mutation as a cause of cortisol-producing adenomas. We aimed to compare the clinical features of PRKACA-mutant lesions with those of CTNNB1 mutations, and to search for similar mutations in unilateral hyperplasia or tumors co-secreting aldosterone. DESIGN, PATIENTS, AND METHODS: In this study, 60 patients with cortisol excess who had adrenalectomies at our institution between 1992 and 2013 were assessed, and somatic mutations were determined by Sanger sequencing. A total of 36 patients had overt Cushing's syndrome, the remainder were subclinical: 59 cases were adenomas (three bilateral) and one was classified as hyperplasia. Four tumors had proven co-secretion of aldosterone. RESULTS: Among cortisol-secreting unilateral lesions without evidence of co-secretion (n=52), we identified somatic mutations in PRKACA (L206R) in 23.1%, CTNNB1 (S45P, S45F) in 23.1%, GNAS (R201C) in 5.8%, and CTNNB1+GNAS (S45P, R201H) in 1.9%. PRKACA and GNAS mutations were mutually exclusive. Of the co-secreting tumors, two (50%) had mutations in KCNJ5 (G151R and L168R). The hyperplastic gland showed a PRKACA(L206R) mutation, while patients with bilateral adenomas did not have known somatic mutations. PRKACA-mutant lesions were associated with younger age, overt Cushing's syndrome, and higher cortisol levels vs non-PRKACA-mutant or CTNNB1-mutant lesions. CTNNB1 mutations were more significantly associated with right than left lesions. CONCLUSIONS: PRKACA(L206R) is present not only in adenomas, but also in unilateral hyperplasia and is associated with more severe autonomous cortisol secretion. Bilateral adenomas may be caused by yet-unknown germline mutations.

Whole-exome sequencing characterizes the landscape of somatic mutations and copy number alterations in adrenocortical carcinoma.

CONTEXT: Adrenocortical carcinoma (ACC) is a rare and lethal malignancy with a poorly defined etiology, and the molecular genetics of ACC are incompletely understood. OBJECTIVE: To utilize whole-exome sequencing for genetic characterization of the underlying somatic mutations and copy number alterations present in ACC. DESIGN: Screening for somatic mutation events and copy number alterations (CNAs) was performed by comparative analysis of tumors and matched normal samples from 41 patients with ACC. RESULTS: In total, 966 nonsynonymous somatic mutations were detected, including 40 tumors with a mean of 16 mutations per sample and one tumor with 314 mutations. Somatic mutations in ACC-associated genes included TP53 (8/41 tumors, 19.5%) and CTNNB1 (4/41, 9.8%). Genes with potential disease-causing mutations included GNAS, NF2, and RB1, and recurrently mutated genes with unknown roles in tumorigenesis comprised CDC27, SCN7A, and SDK1. Recurrent CNAs included amplification at 5p15.33 including TERT (6/41, 14.6%) and homozygous deletion at 22q12.1 including the Wnt repressors ZNRF3 and KREMEN1 (4/41 9.8% and 3/41, 7.3%, respectively). Somatic mutations in ACC-established genes and recurrent ZNRF3 and TERT loci CNAs were mutually exclusive in the majority of cases. Moreover, gene ontology identified Wnt signaling as the most frequently mutated pathway in ACCs. CONCLUSIONS: These findings highlight the importance of Wnt pathway dysregulation in ACC and corroborate the finding of homozygous deletion of Wnt repressors ZNRF3 and KREMEN1. Overall, mutations in either TP53 or CTNNB1 as well as focal CNAs at the ZNRF3 or TERT loci denote mutually exclusive events, suggesting separate mechanisms underlying the development of these tumors.

Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors.

Adrenal tumors autonomously producing cortisol cause Cushing's syndrome. We performed exome sequencing of 25 tumor-normal pairs and identified 2 subgroups. Eight tumors (including three carcinomas) had many somatic copy number variants (CNVs) with frequent deletion of CDC42 and CDKN2A, amplification of 5q31.2 and protein-altering mutations in TP53 and RB1. Seventeen tumors (all adenomas) had no somatic CNVs or TP53 or RB1 mutations. Six of these had known gain-of-function mutations in CTNNB1 (ß-catenin) or GNAS (Gαs). Six others had somatic mutations in PRKACA (protein kinase A (PKA) catalytic subunit) resulting in a p.Leu206Arg substitution. Further sequencing identified this mutation in 13 of 63 tumors (35% of adenomas with overt Cushing's syndrome). PRKACA, GNAS and CTNNB1 mutations were mutually exclusive. Leu206 directly interacts with the regulatory subunit of PKA, PRKAR1A. Leu206Arg PRKACA loses PRKAR1A binding, increasing the phosphorylation of downstream targets. PKA activity induces cortisol production and cell proliferation, providing a mechanism for tumor development. These findings define distinct mechanisms underlying adrenal cortisol-producing tumors.