Kisspeptin deficiency leads to abnormal adrenal glands and excess steroid hormone secretion.

Knockout mice for the kisspeptin receptor, Kiss1r (Kiss1r-/-) and its ligand kisspeptin, Kiss1 (Kiss1-/-) replicate the phenotype of isolated hypogonadotropic hypogonadism (IHH) associated with variants of these genes in humans. A recent report suggests that kisspeptin may be involved in human fetal adrenocortical development and function. Herein, we characterized the adrenal function and morphology in Kiss1-/- mice that do not go through normal puberty. Two fetal markers were expressed in eosinophilic cells potentially derived from the X-zone that should disappear at puberty in male mice and during the first pregnancy in female animals. Although the hypercorticosteronism observed in Kiss1-/- females corrected overtime, hyperaldosteronism persisted at 14 months and correlated with the overexpression of Star. To determine if KISS1 and KISS1R genes are involved in the development of primary aldosteronism (PA) and hypercortisolism [Cushing's syndrome (CS)] in humans, we sequenced these 2 genes in 65 patients with PA and/or CS. Interestingly, a patient with CS presented with a germline KISS1 variant (p.H90D, rs201073751). We also found three rare variants in the KISS1R gene in three patients with PA: p.C95W (rs141767649), p.A189T (rs73507527) and p.R229R (rs115335009). The two missense variants have been previously associated with IHH. Our findings suggest that KISS1 may play a role in adrenal function in mice and possibly adrenocortical steroid hormone secretion in humans, beyond its recently described role in human fetal adrenocortical development.

KDM1A inactivation causes hereditary food-dependent Cushing syndrome.

PURPOSE: This study aimed to investigate the genetic cause of food-dependent Cushing syndrome (FDCS) observed in patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) and adrenal ectopic expression of the glucose-dependent insulinotropic polypeptide receptor. Germline ARMC5 alterations have been reported in about 25% of PBMAH index cases but are absent in patients with FDCS. METHODS: A multiomics analysis of PBMAH tissues from 36 patients treated by adrenalectomy was performed (RNA sequencing, single-nucleotide variant array, methylome, miRNome, exome sequencing). RESULTS: The integrative analysis revealed 3 molecular groups with different clinical features, namely G1, comprising 16 patients with ARMC5 inactivating variants; G2, comprising 6 patients with FDCS with glucose-dependent insulinotropic polypeptide receptor ectopic expression; and G3, comprising 14 patients with a less severe phenotype. Exome sequencing revealed germline truncating variants of KDM1A in 5 G2 patients, constantly associated with a somatic loss of the KDM1A wild-type allele on 1p, leading to a loss of KDM1A expression both at messenger RNA and protein levels (P = 1.2 × 10-12 and P < .01, respectively). Subsequently, KDM1A pathogenic variants were identified in 4 of 4 additional index cases with FDCS. CONCLUSION: KDM1A inactivation explains about 90% of FDCS PBMAH. Genetic screening for ARMC5 and KDM1A can now be offered for most PBMAH operated patients and their families, opening the way to earlier diagnosis and improved management.

Steroidogenic differentiation and PKA signaling are programmed by histone methyltransferase EZH2 in the adrenal cortex.

Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endocrine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phosphodiesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state.

Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

SUMOylation is a highly conserved and dynamic post-translational mechanism primarily affecting nuclear programs for adapting organisms to stressful challenges. Alteration of SUMOylation cycles leads to severe developmental and homeostatic defects and malignancy, but signals coordinating SUMOylation are still unidentified. The adrenal cortex is a zonated endocrine gland that controls body homeostasis and stress response. Here, we show that in human and in mouse adrenals, SUMOylation follows a decreasing centripetal gradient that mirrors cortical differentiation flow and delimits highly and weakly SUMOylated steroidogenic compartments, overlapping glomerulosa, and fasciculata zones. Activation of PKA signaling by acute hormonal treatment, mouse genetic engineering, or in Carney complex results in repression of small ubiquitin-like modifier (SUMO) conjugation in the inner cortex by coordinating expression of SUMO pathway inducers and repressors. Conversely, genetic activation of canonical wingless-related integration site signaling maintains high SUMOylation potential in the outer neoplastic cortex. Thus, SUMOylation is tightly regulated by signaling pathways that orchestrate adrenal zonation and diseases.-Dumontet, T., Sahut-Barnola, I., Dufour, D., Lefrançois-Martinez, A.-M., Berthon, A., Montanier, N., Ragazzon, B., Djari, C., Pointud, J.-C., Roucher-Boulez, F., Batisse-Lignier, M., Tauveron, I., Bertherat, J., Val, P., Martinez, A. Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

Update of Genetic and Molecular Causes of Adrenocortical Hyperplasias Causing Cushing Syndrome.

Bilateral hyperplasias of the adrenal cortex are rare causes of chronic endogenous hypercortisolemia also called Cushing syndrome. These hyperplasias have been classified in two categories based on the adrenal nodule size: the micronodular types include Primary Pigmented Nodular Adrenocortical Disease (PPNAD) and isolated Micronodular Adrenal Disease (iMAD) and the macronodular also named Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH). This review discusses the genetic and molecular causes of these different forms of hyperplasia that involve mutations and dysregulation of various regulators of the cAMP/protein kinase A (PKA) pathway. PKA signaling is the main pathway controlling cortisol secretion in adrenocortical cells under ACTH stimulation. Although mutations of the regulatory subunit R1α of PKA (PRKAR1A) is the main cause of familial and sporadic PPNAD, inactivation of two cAMP-binding phosphodiesterases (PDE11A and PDE8B) are associated with iMAD even if they are also found in PPNAD and PBMAH cases. Interestingly, PBMAH that is observed in multiple familial syndrome such as APC, menin, fumarate hydratase genes, has initially been associated with the aberrant expression of G-protein coupled receptors (GPCR) leading to an activation of cAMP/PKA pathway. However, more recently, the discovery of germline mutations in Armadillo repeat containing protein 5 (ARMC5) gene in 25-50% of PBMAH patients highlights its importance in the development of PBMAH. The potential relationship between ARMC5 mutations and aberrant GPCR expression is discussed as well as the potential other causes of PBMAH.

EZH2 is overexpressed in adrenocortical carcinoma and is associated with disease progression.

Adrenal Cortex Carcinoma (ACC) is an aggressive tumour with poor prognosis. Common alterations in patients include constitutive WNT/ß-catenin signalling and overexpression of the growth factor IGF2. However, the combination of both alterations in transgenic mice is not sufficient to trigger malignant tumour progression, suggesting that other alterations are required to allow development of carcinomas. Here, we have conducted a study of publicly available gene expression data from three cohorts of ACC patients to identify relevant alterations. Our data show that the histone methyltransferase EZH2 is overexpressed in ACC in the three cohorts. This overexpression is the result of deregulated P53/RB/E2F pathway activity and is associated with increased proliferation and poorer prognosis in patients. Inhibition of EZH2 by RNA interference or pharmacological treatment with DZNep inhibits cellular growth, wound healing and clonogenic growth and induces apoptosis of H295R cells in culture. Further growth inhibition is obtained when DZNep is combined with mitotane, the gold-standard treatment for ACC. Altogether, these observations suggest that overexpression of EZH2 is associated with aggressive progression and may constitute an interesting therapeutic target in the context of ACC.

Successful Treatment of Estrogen Excess in Primary Bilateral Macronodular Adrenocortical Hyperplasia with Leuprolide Acetate.

Primary bilateral macronodular adrenocortical hyperplasia (PBMAH) is an uncommon cause of adrenal Cushing syndrome (CS) in which cortisol and occasionally other steroid hormones can be secreted under the influence of aberrantly expressed G-protein coupled receptors (GPCRs) in the adrenal cortex. We describe the unique case of a 64-year-old postmenopausal female with PBMAH whose adrenal lesions expressed luteinizing hormone receptors (LHr). She presented initially with CS and underwent right adrenalectomy; a few years later she presented with macromastia and mastodynia, possibly due to estrogen excess from her remaining left adrenocortical masses. Testing before and after treatment with quarterly leuprolide acetate therapy and immunohistochemistry on tissue and targeted sequencing of the genes of interest were performed. Tissue from the patient's right adrenal was tested for P450 aromatase (CYP19A1) and LHr expression; both were expressed throughout the hyperplastic cortex, although expression was more intense in the adenomatous areas. Targeted sequencing revealed a pathogenic PDE11A mutation, as well as variants in the ARMC5 and INHA genes. PDE11A expression was decreased in the adenoma but there was no loss of heterozygosity for the PDE11A locus. Because of the clinical presentation and LHr expression, quarterly leuprolide acetate therapy was started. Shortly after initiation of therapy, the patient reported decreased breast size and pain; she remains well controlled to date, after 10 years of treatment. This is the first description of a patient with PBMAH presenting with severe macromastia and mastodynia from what appears to be excess estrogen production from her adrenal tumor. The patient had a long-lasting response to chronic leuprolide acetate treatment, showing that drug therapy exploiting the aberrant receptor expression in PBMAH is possible even in the absence of cortisol overproduction.

WNT/ß-catenin signalling is activated in aldosterone-producing adenomas and controls aldosterone production.

Primary aldosteronism (PA) is the main cause of secondary hypertension, resulting from adrenal aldosterone-producing adenomas (APA) or bilateral hyperplasia. Here, we show that constitutive activation of WNT/ß-catenin signalling is the most frequent molecular alteration found in 70% of APA. We provide evidence that decreased expression of the WNT inhibitor SFRP2 may be contributing to deregulated WNT signalling and APA development in patients. This is supported by the demonstration that mice with genetic ablation of Sfrp2 have increased aldosterone production and ectopic differentiation of zona glomerulosa cells. We further show that ß-catenin plays an essential role in the control of basal and Angiotensin II-induced aldosterone secretion, by activating AT1R, CYP21 and CYP11B2 transcription. This relies on both LEF/TCF-dependent activation of AT1R and CYP21 regulatory regions and indirect activation of CYP21 and CYP11B2 promoters, through increased expression of the nuclear receptors NURR1 and NUR77. Altogether, these data show that aberrant WNT/ß-catenin activation is associated with APA development and suggest that WNT pathway may be a good therapeutic target in PA.

Diagnosis and Management of Hereditary Adrenal Cancer.

Benign adrenocortical tumours (ACT) are relatively frequent lesions; on the contrary, adrenocortical carcinoma (ACC) is a rare and aggressive malignancy with unfavourable prognosis. Recent advances in the molecular understanding of adrenal cancer offer promise for better therapies in the future. Many of these advances stem from the molecular elucidation of genetic conditions predisposing to the development of ACC. Six main clinical syndromes have been described to be associated with hereditary adrenal cancer. In these conditions, genetic counselling plays an important role for the early detection and follow-up of the patients and the affected family members.

Impact of Morphology in the Genotype and Phenotype Correlation of Bilateral Macronodular Adrenocortical Disease (BMAD): A Series of Clinicopathologically Well-Characterized 35 Cases.

Bilateral macronodular adrenocortical disease (BMAD) is characterized by the development of adrenal macronodules resulting in a pituitary-ACTH independent Cushing's syndrome. Although there are important similarities observed between the rare microscopic descriptions of this disease, the small series published are not representative of the molecular and genetic heterogenicity recently described in BMAD. We analyzed the pathological features in a series of BMAD and determined if there is correlation between these criteria and the characteristics of the patients. Two pathologists reviewed the slides of 35 patients who underwent surgery for suspicion of BMAD in our center between 1998 and 2021. An unsupervised multiple factor analysis based on microscopic characteristics divided the cases into 4 subtypes according to the architecture of the macronodules (containing or not round fibrous septa) and the proportion of the different cell types: clear, eosinophilic compact, and oncocytic cells. The correlation study with genetic revealed subtype 1 and subtype 2 are associated with the presence of ARMC5 and KDM1A pathogenic variants, respectively. By immunohistochemistry, all cell types expressed CYP11B1 and HSD3B1. HSD3B2 staining was predominantly expressed by clear cells whereas CYP17A1 staining was predominant on compact eosinophilic cells. This partial expression of steroidogenic enzymes may explain the low efficiency of cortisol production in BMAD. In subtype 1, trabeculae of eosinophilic cylindrical cells expressed DAB2 but not CYP11B2. In subtype 2, KDM1A expression was weaker in nodule cells than in normal adrenal cells; alpha inhibin expression was strong in compact cells. This first microscopic description of a series of 35 BMAD reveals the existence of 4 histopathological subtypes, 2 of which are strongly correlated with the presence of known germline genetic alterations. This classification emphasizes that BMAD has heterogeneous pathological characteristics that correlate with some genetic alterations identified in patients.

Constitutive beta-catenin activation induces adrenal hyperplasia and promotes adrenal cancer development.

Adrenocortical carcinoma is a rare but aggressive cancer with unknown aetiology. Constitutive activation of beta-catenin is the most frequent alteration in benign and malignant adrenocortical tumours in patients. Here, we show that constitutive activation of beta-catenin in the adrenal cortex of transgenic mice resulted in progressive steroidogenic and undifferentiated spindle-shaped cells hyperplasia as well as dysplasia of the cortex and medulla. Over a 17 months time course, transgenic adrenals developed malignant characteristics such as uncontrolled neovascularization and loco-regional metastatic invasion. These oncogenic events were accompanied by ectopic differentiation of glomerulosa at the expense of fasciculata cells, which caused primary hyperaldosteronism. Altogether these observations demonstrate that constitutively active beta-catenin is an adrenal oncogene which triggers benign aldosterone-secreting tumour development and promotes malignancy.

Primary bilateral macronodular adrenal hyperplasia: definitely a genetic disease.

Primary bilateral macronodular adrenal hyperplasia (PBMAH) is an adrenal cause of Cushing syndrome. Nowadays, a PBMAH diagnosis is more frequent than previously, as a result of progress in the diagnostic methods for adrenal incidentalomas, which are widely available. Although some rare syndromic forms of PBMAH are known to be of genetic origin, non-syndromic forms of PBMAH have only been recognized as a genetic disease in the past 10 years. Genomics studies have highlighted the molecular heterogeneity of PBMAH and identified molecular subgroups, allowing improved understanding of the clinical heterogeneity of this disease. Furthermore, the generation of these subgroups permitted the identification of new genes responsible for PBMAH. Constitutive inactivating variants in ARMC5 and KDM1A are responsible for the development of distinct forms of PBMAH. To date, pathogenic variants of ARMC5 are responsible for 20-25% of PBMAH, whereas germline KDM1A alterations have been identified in >90% of PBMAH causing food-dependent Cushing syndrome. The identification of pathogenic variants in ARMC5 and KDM1A demonstrated that PBMAH, despite mostly being diagnosed in adults aged 45-60 years, is a genetic disorder. This Review summarizes the important progress made in the past 10 years in understanding the genetics of PBMAH, which have led to a better understanding of the pathophysiology, opening new clinical perspectives.

Tumor suppressor gene ARMC5 controls adrenal redox state through NRF1 turnover.

ARMC5: is a tumor suppressor gene frequently mutated in primary bilateral macronodular adrenal hyperplasia (PBMAH), an adrenal cause of Cushing's syndrome. The function of ARMC5 is poorly understood, aside from the fact that it regulates cell viability and adrenal steroidogenesis by mechanisms still unknown. Tumor suppressor genes play an important role in modifying intracellular redox response, which in turn regulates diverse cell signaling pathways. In this study, we demonstrated that inactivation in adrenocortical cells increased the expression of actors scavenging reactive oxygen species, such as superoxide dismutases (SOD) and peroxiredoxins (PRDX) by increasing the transcriptional regulator NRF1. Moreover, ARMC5 is involved in the NRF1 ubiquitination and in its half-life. Finally, inactivation alters adrenocortical steroidogenesis through the activation of p38 pathway and decreases cell sensitivity to ferroptosis participation to increase cell viability. Altogether, this study uncovers a function of ARMC5 as a regulator of redox homeostasis in adrenocortical cells, controlling steroidogenesis and cell survival.

Insulin-like growth factor 2 (IGF2) expression in adrenocortical disease due to PRKAR1A mutations compared to other benign adrenal tumors.

PURPOSE: Insulin-like growth factor-II (IGF2), a key regulator of cell growth and development, is tightly regulated in its expression by epigenetic control that maintains its monoallelic expression in most tissues. Biallelic expression of IGF2 resulting from loss of imprinting (LOI) has been reported in adrenocortical tumors. In this study, we wanted to check whether adrenocortical lesions due to PRKAR1A mutations lead to increased IGF2 expression from LOI and compare these findings to those in other benign adrenal lesions. METHODS: We compared the expression of IGF2 by RNA and protein studies in primary pigmented nodular adrenocortical disease (PPNAD) caused by PRKAR1A gene mutations to that in primary macronodular adrenocortical hyperplasia (PMAH) and cortisol-producing adenomas (CPA) that did not have any mutations in known genes. We also checked LOI in all lesions by DNA allelic studies and the expression of other components of IGF2 signaling at the RNA and protein level. RESULTS: We identified cell clusters overexpressing IGF2 in PPNAD; although immunostaining was patchy, overall, by RNA and immunoblotting PPNAD expressed high IGF2 message and protein. However, this was not due to LOI, as there was no correlation between IGF2 expression and the presence of LOI. CONCLUSIONS: Our data pointed to over-expression of IGF2 protein in PPNAD compared to other benign adrenocortical lesions, such as PMAH and CPA. However, there was no correlation of IGF2 mRNA levels with LOI of IGF2/H19. The discrepancy between mRNA and protein levels with regards to LOI points, perhaps, to different control of IGF2 gene expression in PPNAD.

Pde8b haploinsufficiency in mice is associated with modest adrenal defects, impaired steroidogenesis, and male infertility, unaltered by concurrent PKA or Wnt activation.

PDE8B, PRKAR1A and the Wnt/ß-catenin signaling are involved in endocrine disorders. However, how PDEB8B interacts with both Wnt and protein kinase A (PKA) signaling in vivo remains unknown. We created a novel Pde8b knockout mouse line (Pde8b-/-); Pde8b haploinsufficient (Pde8b+/-) mice were then crossed with mice harboring: (1) constitutive beta-catenin activation (Pde8b+/-;ΔCat) and (2) Prkar1a haploinsufficieny (Pde8b+/-;Prkar1a+/-). Adrenals and testes from mice (3-12-mo) were evaluated in addition to plasma corticosterone, aldosterone and Dkk3 concentrations, and the examination of expression of steroidogenesis-, Wnt- and cAMP/PKA-related genes. Pde8b-/- male mice were infertile with down-regulation of the Wnt/ß-catenin pathway which did not change significantly in the Pde8b+/-;ΔCat mice. Prkar1a haploinsufficiency also did not change the phenotype significantly. In vitro studies showed that PDE8B knockdown upregulated the Wnt pathway and increased proliferation in CTNNB1-mutant cells, whereas it downregulated the Wnt pathway in PRKAR1A-mutant cells. These data support an overall weak, if any, role for PDE8B in adrenocortical tumorigenesis, even when co-altered with Wnt signaling or PKA upregulation; on the other hand, PDE8B appears to play a significant role in male fertility.

Inhibition of Aurora kinase A activity enhances the antitumor response of beta-catenin blockade in human adrenocortical cancer cells.

Adrenocortical cancer (ACC) is a rare and aggressive type of endocrine tumor with high risk of recurrence and metastasis. The overall survival of patients diagnosed with ACC is low and treatment for metastatic stages remain limited to mitotane, which has low efficiency in advanced stages of the disease and is associated with high toxicity. Therefore, identification of new biological targets to improve ACC treatment is crucial. Blockade of the Wnt/beta-catenin pathway decreased adrenal steroidogenesis and increased apoptosis of NCI-H295 human ACC cells, in vitro and in a xenograft mouse model. Aurora kinases play important roles in cell division during the G1-M phase and their aberrant expression is correlated with a poor prognosis in different types of tumors. Hence, we hypothesized that inhibition of aurora kinases activity combined with the beta-catenin pathway blockade would improve the impairment of ACC cell growth in vitro. We studied the combinatorial effects of AMG 900, an aurora kinase inhibitor and PNU-74654, a beta-catenin pathway blocker, on proliferation, survival and tumor progression in multiple ACC cell lines: NCI-H295, CU-ACC1 and CU-ACC2. Exposure of ACC cells to the combination of AMG 900 with PNU-74654 decreased cell proliferation and viability compared to either treatment alone. In addition, AMG 900 inhibited cell invasion and clonogenesis compared to PNU-74654, and the combination showed no greater effects. In contrast, PNU-74654 was more effective in decreasing cortisol secretion. These data suggest that inhibition of aurora kinases activity combined with blockade of the beta-catenin pathway may provide a combinatorial approach for targeting ACC tumors.

Volumetric Modeling of Adrenal Gland Size in Primary Bilateral Macronodular Adrenocortical Hyperplasia.

CONTEXT: Radiological characterization of adrenal size in primary bilateral macronodular adrenocortical hyperplasia (PBMAH) has not been previously investigated. OBJECTIVE: We hypothesized that volumetric modeling of adrenal gland size may correlate with biochemical disease severity in patients with PBMAH. Secondary analysis of patients with concurrent primary aldosteronism (PA) was performed. DESIGN: A retrospective cross-sectional analysis of 44 patients with PBMAH was conducted from 2000 to 2019. SETTING: Tertiary care clinical research center. PATIENTS: Patients were diagnosed with PBMAH based upon clinical, genetic, radiographic and biochemical characteristics. INTERVENTION: Clinical, biochemical, and genetic data were obtained. Computed tomography scans were used to create volumetric models by manually contouring both adrenal glands in each slice using Vitrea Core Fx v6.3 software (Vital Images, Minnetonka, Minnesota). MAIN OUTCOME AND MEASURES: 17-hydroxycorticosteroids (17-OHS), ARMC5 genetics, and aldosterone-to-renin ratio (ARR) were retrospectively obtained. Pearson test was used for correlation analysis of biochemical data with adrenal volume. RESULTS: A cohort of 44 patients with PBMAH was evaluated, with a mean age (±SD) of 53 ±â€…11.53. Eight patients met the diagnostic criteria for PA, of whom 6 (75%) were Black. In the Black cohort, total adrenal volumes positively correlated with midnight cortisol (R = 0.76, P = 0.028), urinary free cortisol (R = 0.70, P = 0.035), and 17-OHS (R = 0.87, P = 0.0045), with a more pronounced correlation with left adrenal volume alone. 17-OHS concentration positively correlated with total, left, and right adrenal volume in patients harboring pathogenic variants in ARMC5 (R = 0.72, P = 0.018; R = 0.65, P = 0.042; and R = 0.73, P = 0.016, respectively). CONCLUSIONS: Volumetric modeling of adrenal gland size may associate with biochemical severity in patients with PBMAH, with particular utility in Black patients.

ARMC5 variants in PRKAR1A-mutated patients modify cortisol levels and Cushing's syndrome.

Mutations in the protein kinase A (PKA) regulatory subunit type 1A (PRKAR1A) and armadillo repeat-containing 5 (ARMC5) genes cause Cushing's syndrome (CS) due to primary pigmented nodular adrenocortical disease (PPNAD) and primary bilateral macronodular adrenocortical hyperplasia (PBMAH), respectively. Between the two genes, ARMC5 is highly polymorphic with several variants in the population, whereas PRKAR1A has very little, if any, non-pathogenic variation in its coding sequence. We tested the hypothesis that ARMC5 variants may affect the clinical presentation of PPNAD and CS among patients with PRKAR1A mutations. In this study, 91 patients with PPNAD due to PRKAR1A mutations were tested for abnormal cortisol secretion or CS and for ARMC5 sequence variants. Abnormal cortisol secretion was present in 71 of 74 patients with ARMC5 variants, whereas 11 of 17 patients negative for ARMC5 variants did not have hypercortisolemia. The presence of ARMC5 variants was a statistically strong predictor of CS among patients with PRKAR1A mutations (P < 0.001). Among patients with CS due to PPNAD, ARMC5 variants were associated with lower cortisol levels at baseline (P = 0.04) and after high dose dexamethasone administration (P = 0.02). The ARMC5 p.I170V variant increased ARMC5 protein accumulation in vitro and decreased viability of NCI-H295 cells (but not HEK 293T cells). PPNAD tissues with ARMC5 variants showed stronger ARMC5 protein expression than those that carried a normal ARMC5 sequence. Taken together, our results suggest that ARMC5 variants among patients with PPNAD due to PRKAR1A defects may play the role of a genetic modifier for the presence and severity of hypercortisolemia.

The Association of ARMC5 with the Renin-Angiotensin-Aldosterone System, Blood Pressure, and Glycemia in African Americans.

CONTEXT: Armadillo repeat containing 5 (ARMC5) on chromosome 16 is an adrenal gland tumor suppressor gene associated with primary aldosteronism, especially among African Americans (AAs). We examined the association of ARMC5 variants with aldosterone, plasma renin activity (PRA), blood pressure, glucose, and glycosylated hemoglobin A1c (HbA1c) in community-dwelling AAs. METHODS: The Jackson Heart Study is a prospective cardiovascular cohort study in AAs with baseline data collection from 2000 to 2004. Kernel machine method was used to perform a single joint test to analyze for an overall association between the phenotypes of interest (aldosterone, PRA, systolic and diastolic blood pressure [SBP, DBP], glucose, and HbA1c) and the ARMC5 single nucleotide variants (SNVs) adjusted for age, sex, BMI, and medications; followed by Baysian Lasso methodology to identify sets of SNVs in terms of associated haplotypes with specific phenotypes. RESULTS: Among 3223 participants (62% female; mean age 55.6 (SD ± 12.8) years), the average SBP and DBP were 127 and 76 mmHg, respectively. The average fasting plasma glucose and HbA1c were 101 mg/dL and 6.0%, respectively. ARMC5 variants were associated with all 6 phenotypes. Haplotype TCGCC (ch16:31476015-31476093) was negatively associated, whereas haplotype CCCCTTGCG (ch16:31477195-31477460) was positively associated with SBP, DBP, and glucose. Haplotypes GGACG (ch16:31477790-31478013) and ACGCG (ch16:31477834-31478113) were negatively associated with aldosterone and positively associated with HbA1c and glucose, respectively. Haplotype GCGCGAGC (ch16:31471193-ch16:31473597(rs114871627) was positively associated with PRA and negatively associated with HbA1c. CONCLUSIONS: ARMC5 variants are associated with aldosterone, PRA, blood pressure, fasting glucose, and HbA1c in community-dwelling AAs, suggesting that germline mutations in ARMC5 may underlie cardiometabolic disease in AAs.