Armadillo Repeat Containing 5 Research Articles

Prevalence and clinical features of armadillo repeat-containing 5 mutations carriers in a single center cohort of patients with bilateral adrenal incidentalomas.

We aimed to evaluate the prevalence of armadillo repeat-containing 5 (ARMC5) genetic defects in our cohort of bilateral adrenal incidentaloma (BAI) patients and to evaluate the possible existence of genotype-phenotype correlations. Cross-sectional study. Tertiary care center. 72 BAI patients. The following data have been collected: morning adrenocorticotropic hormone (ACTH) concentrations; cortisol levels after 1 mg overnight dexamethasone suppression test (F-1mgDST); urinary free cortisol (UFC) levels; diameter of the adrenal masses; and the association with overweight/obesity, arterial hypertension, diabetes mellitus, dyslipidemia, cardiovascular events, unrelated neoplasia, osteoporosis, thyroid nodular disease, and primary hyperparathyroidism. A search for ARMC5 germline and somatic pathogenic variants was performed in all patients and in the adrenal tissue of patients operated on, respectively. The prevalence of germline ARMC5 pathogenic variants among patients with mild autonomous cortisol secretion (MACS+, defined as F-1mgDST > 1.8 µg/dL) was 18.8%. No germline pathogenic variants were detected in patients without MACS. Moreover, somatic ARMC5 pathogenic variants were also found in the adrenal tissue of six patients without germline ARMC5 variants. The F-1mgDST levels >5 µg/dL predicted with a poor sensitivity but a 90.5% specificity in identifying the presence of ARMC5 germline pathogenic variants. We did not find any clinical parameter predictive of the ARMC5 mutation presence. In MACS+ BAI patients, germline ARMC5 gene pathogenic variants are frequent. Further studies are needed to elucidate the pathophysiological role of somatic ARMC5 pathogenic variants on adrenal tumor development in otherwise wild-type (WT) patients.

Morphological Harbingers of ARMC5-Pathogenic Variant-Related Bilateral Macronodular Adrenocortical Disease.

Bilateral macronodular adrenocortical disease (BMAD) is a neoplastic disease associated with a high frequency of germline disease. Armadillo repeat containing 5 (ARMC5) pathogenic variants (PVs) have not been widely studied to determine the morphological and immunohistochemical characteristics of BMAD. We carried out a detailed morphologic review of 22 surgical specimens excised from patients with BMAD and compared them with PV of ARMC5 (PV + , n = 14) and those without (PV - , n = 8), and further comparing them with a control group of adrenals excised from patients with renal cancer (n = 11). No patients presented with a genetic syndrome related to BMAD. Overt Cushing's syndrome was present in 12/22 patients, 10 PV + and 2 PV - (p = 0.074). We also evaluated the expression of Ki-67, BCL-2, BAX, p53, CYP11B1, and ARMC5 protein. The pseudo-glandular and trabecular architectural patterns were strongly associated with the PV + group (both p < 0.001), as well as capsular extrusion (p < 0.001). There was no predictive value in the distinction of ARMC5 variants in Hsiao subtyping. ARMC5 diffuse cytoplasmic staining was observed in all 11 control samples. The ARMC5 expression was significantly lower in BMAD than in the control group (p < 0.001). In all the specimens, expression of BCL-2 was identified only in the medulla, and expression of BAX was observed in adrenocortical cells. CYP11B1 diffuse immunoexpression was identified in all the specimens of BMAD and in the fasciculata zone in the control group. The mitotic count and Ki-67 proliferation index was very low in all three groups (controls, PV + , and PV - BMAD). None of the specimens stained positive for the p53 protein. Although our series is limited, the presence of pseudo-glandular and/or trabecular patterns and capsular extrusion indicated the presence of pathogenic variants of ARMC5 in BMAD. The gland enlargement does not seem to be related to the increase of mitotic count or a higher proliferation index (Ki-67).

A novel pathogenic variant of ARMC5 in a patient with primary bilateral macronodular adrenal hyperplasia: a case report

BackgroundPrimary bilateral macronodular adrenal hyperplasia (PBMAH), also known as adrenocorticotropic hormone (ACTH)-independent macronodular adrenal hyperplasia, is a rare cause of endogenous Cushing’s syndrome. In many familial cases of PBMAH, the variants in armadillo repeat containing 5 (ARMC5) gene are found to be associated with the disease. Here, we report a case of PBMAH harboring a novel frameshift variant in ARMC5 gene, which has not been previously reported in the literature.Case presentationA 67-year-old woman was referred due to the clinical features of Cushing’s syndrome. Radiological imaging and hormonal testing were carried out. The serum levels of cortisol were remarkably increased at late night and did not suppress even after 1 mg of dexamethasone administration, while the plasma levels of ACTH hormone were decreased significantly. The patient underwent unilateral left-sided laparoscopic adrenalectomy, and the diagnosis of PBMAH was substantiated by histopathological analysis. Moreover, the partial envelope was incomplete and the cell proliferation index was low. Specifically, inhibin α-subunit ( +), syn focal ( +), Ki-67 ~ 3% ( +), CgA (-) and CEA (-) were observed. DNA sequencing data revealed that a novel frameshift variant (c.363_373delGCCAGTGCGCC, p.Pro122Alafs*61) was identified in ARMC5 gene. However, this variant was not detected in the daughter of the patient. The rest of the family members, including her sister, son and two brothers, were not consented for genetic testing.ConclusionsEarly detection of ARMC5 variant status and familial screening might have important clinical implications for the diagnosis and prognosis of PBMAH patients. A novel ARMC5 frameshift variant (c.363_373delGCCAGTGCGCC, p.Pro122Alafs*61) was identified to be associated with the pathogenesis of PBMAH. ARMC5 sequencing may improve the identification of a causative gene variant for PBMAH and allow earlier diagnosis of this disease.

Follow-up of second adrenal tumor after remission of Cushing syndrome

Bilateral adrenal tumors (BAT) represent a vast domain of endocrinology and connected medical and surgical fields. Our purpose is to introduce several key points in relationship with long time management on a 56-year old female case who is currently admitted for reassessment of a left adrenal tumor with potential autonomous cortisol secretion. She is also known with pituitary incidentaloma since 2014, cerebral meningioma which was partially removed in 2015, uncontrolled diabetes mellitus under metformin and insulin therapy since 2017, and high blood pressure since 2021. Her medical history includes right adrenalectomy for Cushing syndrome in 2014. At that moment, she was first admitted for BAT, a tumor of 2 centimeters (cm) on the left gland according to computed tomography, respective of 3.5 cm maximum diameter on the right adrenal. The hormonal panel confirmed adrenal Cushing syndrome. After 6 months of non-interventional follow-up, the right tumor increased to 4 cm, also associating small areas of necrosis, thus a decision of unilateral laparoscopic adrenalectomy was done with good clinical post-operatory outcome. She did not develop adrenal insufficiency at any point in time, moreover, a low-normal ACTH (Adrenocorticotropic Hormone) with intermittent elevation of morning plasma cortisol levels after Dexamethasone suppression test showed a possible autonomous cortisol secretion of the left adrenal tumor during a 7-year follow-up. In 2014, the female patient received the confirmation of an adrenocortical adenoma which was consistent with cortisol over-secretion. Particular gene contributions are attributed to protein kinase A (PKA) defects (or B) that usually induce bilateral adrenocortical hyperplasia; however, they are not routinely tested in daily endocrine practice, neither had we performed it. Interestingly, the patient was detected with a cerebral meningioma after years of intermittent, mild headache. The diagnostic was established during initial endocrine evaluations. The headache was not associated with uncontrolled high blood pressure, as first expected due to excessive cortisol amount. Recently, it was identified that mutations of ARMC5 (armadillo repeat containing 5) gene are responsible for macronodular adrenal hyperplasia, but also meningioma. They may be also responsible for severe diabetes mellitus as seen here. The key points of following a patient with bilateral adrenal tumors include the timing of uni/bilateral adrenalectomy, the multidisciplinary management of associated complications, as well as the need of understanding the genetic rational behind it.

Primary Macronodular Adrenal Hyperplasia Associated With Autosomal Dominant ARMC5 Mutation

Background: Primary macronodular adrenal hyperplasia (PMAH) is an uncommon cause of Cushing’s syndrome. In some cases, this is an inherited disorder due to a mutation in the armadillo repeat-containing 5 (ARMC5) gene. Clinical Case: A 43-year-old African American woman presented to clinic with weight gain, worsening type 2 diabetes mellitus, and symptomatic hypertensive cardiomyopathy. Physical exam was significant for central obesity, bilateral supraclavicular fat pads, acanthosis nigricans and wide striae over the abdomen. Her serum cortisol was 13.8 mcg/dL after 1 mg of dexamethasone (n<1.8 mcg/dL), urinary cortisol was 180 mcg in 24 hours (n=3.5–45 mcg/24h) and two midnight salivary cortisol tests were 227 and 118 ng/dL (n<100 ng/dL). Her ACTH was 2.4 pg/mL (n=7.2–63.3 pg/mL). Computed tomography (CT) of the abdomen showed nodularity, diffuse thickening and low-density (<10 Hounsfield units) of the bilateral adrenal glands. She underwent bilateral adrenalectomy for a diagnosis of PMAH. Pathology showed nodular adrenocortical hyperplasia; the right and left adrenal glands measured 75 grams and 68 grams, respectively. She was started on hydrocortisone and fludrocortisone postoperatively. Over the following two years, she had a 68-pound weight loss, an 86% reduction in her daily insulin requirement and a 10% improvement in her left ventricular ejection fraction. Approximately two years later, the patient’s brother was referred for bilateral macronodular hyperplasia incidentally discovered on a CT of the abdomen. He had a history of hypertension and type 2 diabetes mellitus with cushingoid features on exam. His serum cortisol was 20.7 mcg/dL after 1 mg of dexamethasone, urinary cortisol was 65.1 mcg in 24 hours, two midnight salivary cortisol tests were 232 and 404 ng/dL and ACTH was 2.0 pg/mL. Upon obtaining further family history, the patients reported clinical features of Cushing’s syndrome in their paternal grandmother but denied features in either parent. The second patient had genetic testing which showed a mutation in the ARMC5 gene, c.1777C>T, p.R593W, a mutation previously described. Due to clinical signs of Cushing’s syndrome, he underwent bilateral adrenalectomy in which pathology showed right and left nodular adrenocortical hyperplasia measuring 110 and 73 grams, respectively. He is doing well postoperatively. The patients recently reported their aunt was diagnosed with PMAH and mild Cushing’s syndrome. She had a unilateral adrenalectomy of the larger adrenal gland and is doing well postoperatively. Conclusion: In some cases, Cushing’s syndrome is an inherited disorder. Autosomal dominant mutations in the ARMC5 gene are occasionally seen in PMAH, which causes less than 2% of endogenous Cushing’s syndrome. For all patients diagnosed with PMAH, clinicians should consider screening their family members with a dexamethasone suppression test.

ARMC5 Alterations in Patients With Sporadic Neuroendocrine Tumors and Multiple Endocrine Neoplasia Type 1 (MEN1).

Adrenal lesions are frequent among patients with sporadic neuroendocrine tumors (spNETs) or multiple endocrine neoplasia type 1 (MEN1). Armadillo repeat-containing 5 (ARMC5)-inactivating variants cause adrenal tumors and possibly other neoplasms. The objective of this work is to investigate a large cohort spNETs or MEN1 patients for changes in the ARMC5 gene. A total of 111 patients, 94 with spNET and 17 with MEN1, were screened for ARMC5 germline alterations. Thirty-six tumors (18 spNETs and 18 MEN1 related) were collected from 20 patients. Blood and tumor DNA samples were genotyped using Sanger sequencing and microsatellite markers for chromosomes. ARMC5 and MEN1 expression were assessed by immunohistochemistry. In 76 of 111 (68.4%) patients, we identified 16 different ARMC5 germline variants, 2 predicted as damaging. There were no differences in the prevalence of ARMC5 variants depending on the presence of MEN1-related adrenal lesions. Loss of heterozygosity (LOH) at chromosome 16p and ARMC5 germline variants were present together in 23 or 34 (67.6%) tumors; in 7 of 23 (30.4%) their presence led to biallelic inactivation of the ARMC5 gene. The latter was more prevalent in MEN1-related tumors than in spNETs (88.9% vs 38.9%; P = .005). LOH at the chromosome 16p (ARMC5) and 11q (MEN1) loci coexisted in 16/18 MEN1-related tumors, which also expressed lower ARMC5 (P = .02) and MEN1 (P = .01) proteins compared to peritumorous tissues. Germline ARMC5 variants are common among spNET and MEN1 patients. ARMC5 haploinsufficiency or biallelic inactivation in spNETs and MEN1-related tumors suggests that ARMC5 may have a role in modifying the phenotype of patients with spNETs and/or MEN1 beyond its known role in macronodular adrenocortical hyperplasia.

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.

Approach to patients with bilateral adrenal incidentalomas.

The current review provides a summary on the most recent developments regarding the cause, work-up and management of bilateral adrenal incidentalomas (BAI). The recent ENS@T/ESE guidelines provide comprehensive directions on the evaluation and management of patients with adrenal incidentalomas with special focus on those with bilateral tumours. Intraadrenal ACTH synthesis that may locally stimulate cortisol secretion challenging the traditionally used term 'ACTH-independent'. Inactivating mutations of a new tumour suppressor gene, armadillo repeat containing 5 (ARMC5), are implicated in a number of patients, especially those with multiple macronodules (bilateral macronodular hyperplasia) and evidence of hypercortisolism. Loss-of-function mutations of the glucocorticoid receptor gene (NR3C1) consist a new possible genetic cause of BAIs. Regarding management an increasing number of studies provide data on the benefits and safety of unilateral rather than bilateral adrenalectomy. There is also emerging data on the beneficial use of steroidogenesis inhibitors in a dosing schedule that aims to mimic the normal cortisol rhythm with promising short-term results, but the long-term clinical benefits of this approach remain to be demonstrated. The diagnostic approach consists of imaging and hormonal evaluation. Imaging characterization should be done separately for each lesion. Hormonal evaluation includes testing for primary aldosteronism, pheochromocytoma and evaluation for autonomous cortisol secretion, using the 1-mg overnight dexamethasone suppression test. Midnight cortisol or 24-h urinary-free cortisol may aid in establishing the degree of cortisol excess. In patients with hypercortisolism ACTH levels should be measured to establish ACTH-independency. The appropriate management of BAI associated with cortisol excess remains controversial. Bilateral adrenalectomy results in lifetime steroid dependency and is better reserved only for patients with overt and severe hypercortisolism. Unilateral adrenalectomy might be considered in selected patients. Medical therapy is not an established approach yet but it may be considered when control of hypercortisolism is desired, but surgery is not an option.

SAT-193 Clinical Case of ARMC5 Tumor Syndrome: A Rare Case of Cushing’s Syndrome from Primary Bilateral Macronodular Adrenal Hyperplasia Caused by ARMC5 Mutation with Concomitant Presence of Meningiomas and Primary Hyperparathyroidism

BACKGROUND: Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) is a known rare cause of Cushing’s syndrome (CS). A mutation in the armadillo repeat containing 5 (ARMC5) sequence is associated with up to 55% of PBMAH cases. Recent studies have linked ARMC5 mutations to presence of other benign neoplasias suggesting that ARMC5 could be a tumor suppressor gene. Case: 72-year-old female with a history of obesity, HTN, DM2, osteoporosis, multiple meningiomas, and hypercalcemia with recurrent kidney stones was incidentally found to have bilateral adrenal nodules on CT imaging. She had mild cushingoid features with truncal obesity and moon facies. She had multiple low dose dexamethasone suppression tests with AM cortisol levels in 17-21 ug/dL range (<1.8 ug/dL). She had late night salivary cortisols of 0.885ug/dL and 1.935ug/dL (0.022-0.254 ug/dl in PM). The overall clinical picture of obesity, HTN and DM2 in combination with biochemical testing and presence of bilateral adrenal adenomas was suggestive of CS secondary to PBMAH. On her evaluation for recurrent kidney stones she had a PTH of 107.2 pg/mL (15-65 pg/mL), and a calcium of 10.8 mg/dL (8.3-10.5 mg/dL). She was diagnosed with primary hyperparathyroidism. Imaging studies found a 1.1 cm ectopic parathyroid adenoma situated at the aortic pulmonary window. Surgical evaluation was performed and surgery was not offered given the precarious location of the parathyroid tumor. She had a known history of four meningiomas, two of which were resected and two considered uncresectable. PBMAH in presence of all her other medical comorbidities prompted genetic evaluation for the patient. Analysis revealed a heterozygous ARMC5 mutation. Given the familial pattern of inheritance associated with ARMC5 mutations, patient’s daughter also underwent genetic testing. Daughter tested positive for the mutation as well. Patient was offered surgical and medical therapy options for her PBMAH. She is currently being evaluated for an unilateral adrenalectomy. Discussion: The pathophysiology of CS from PBMAH remains poorly understood leading to an insidious delay in diagnosis and treatment. Inactivating ARMC5 mutations of familial origins are known genetic triggers for development of PBMAH. ARMC5 is also a proposed tumor suppressor gene whose proteins are found in endocrine tissues all over body. Mutation of ARMC5 gene potentially can lead to multi-glandular tumor syndromes. Screening PBMAH patients and their family members for ARMC5 mutations may lead to earlier CS diagnosis/treatment times as well as better understanding of the gene’s neoplastic potential.

SAT-197 Unilateral Adrenalectomy Can Induce Control in ARMC5 Mutations Patients

Primary macronodular hyperplasia (PMAH) is a rare cause of endogenous Cushing’s syndrome characterized by functioning adrenal macronodules and variable cortisol secretion. ARMC5 is the most frequent gene responsible for PMAH. Genetic mutations including inactivating germline mutations in armadillo repeat-containing 5 (ARMC5) gene have been identified. The occurrence of several other non adrenal tumors (meningioma, breast, colon, thyroid and parathyroid) has also been associated with PBMAH, suggesting a possible role of ARMC5 for the development of other neoplasias. The best treatment to this condition is not established. Clinical case: 64-year-old man, referred due to bilateral adrenal incidentalomas on a CT scan with characteristics of adenoma/hyperplasia. He had type-2 diabetes, hypertension and dyslipidemia for about 4 years. He was being treated with 4 different anti-hypertensive medications, gliclazide and simvastatin. Physical examination revealed thin and dry skin, obesity with centripetal fat distribution, multiple ecchymosis and facial erythrosis. He had a son and a daughter. No known familial relevant diseases. His laboratory workup revealed ACTH-independent hypercortisolism: failure to suppress on the overnight dexametasone suppression test - cortisol 27.6ug/dL- and on the low-dose dexamethasone test- cortisol 24ug/dL- associated to morning ACTH <1ng/L. He underwent stimulation tests with tetracosactide, LHRH, TRH, vasopressin, metoclopramide, glucagon anddeambulation test, which were overall negative. It was decided to proceed to bilateral adrenalectomy. Due to surgical complications, only right adenalectomy was performed. Histology revealed adrenal nodular hyperplasia. Molecular study in DNA extracted from peripheral blood leucocyte and in the adrenal gland revealed the presence in heterozygosity of the pathogenic mutation c.1379T>C in the ARMC5 gene. The patient underwent a cerebral CT scan which showed a meningioma in the posterior left temporal convexity. Genetic testing was also performed on the daughter and son of the patient, which revealed the same mutation. They were also tested for hypercortisolism and underwent adrenal and cerebral CT scan, which showed no abnormality. The adrenal CT scan of the daughter showed enlargement of the left adrenal gland, with aspects suggestive of nodular hyperplasia. Her blood tests revealed no sign of hypercortisolism - overnight dexametasone test- cortisol 1.0 ug/dL, morning ACTH 19.1 ng/L. The patient is currently with cortisol hypersecretion controlled. Conclusion: All patients with PMAH should be tested for ARMC5 mutations and if they are found, family screening is mandatory in this autosomal dominant disorder. Unilateral adrenalectomy could control the cortisol hypersecretion, however some of these patients can have subclinical Cushing Syndrome.

Cullin 3 is a partner of armadillo repeat containing 5 (ARMC5), the product of the gene responsible for primary bilateral macronodular adrenal hyperplasia

Cullin 3 is a partner of armadillo repeat containing 5 (ARMC5), the product of the gene responsible for primary bilateral macronodular adrenal hyperplasia

Genetics of Hypertension in African Americans and Others of African Descent.

Hypertension is the leading cause of cardiovascular disease in the United States, affecting up to one-third of adults. When compared to other ethnic or racial groups in the United States, African Americans and other people of African descent show a higher incidence of hypertension and its related comorbidities; however, the genetics of hypertension in these populations has not been studied adequately. Several genes have been identified to play a role in the genetics of hypertension. They include genes regulating the renin-aldosterone-angiotensin system (RAAS), such as Sodium Channel Epithelial 1 Beta Subunit (SCNN1B), Armadillo Repeat Containing 5 (ARMC5), G Protein-Coupled Receptor Kinase 4 (GRK4), and Calcium Voltage-Gated Channel Subunit Alpha1 D (CACNA1D). In this review, we focus on recent genetic findings available in the public domain for potential differences between African Americans and other populations. We also cover some recent and relevant discoveries in the field of low-renin hypertension from our laboratory at the National Institutes of Health. Understanding the different genetics of hypertension among various groups is essential for effective precision-guided medical therapy of high blood pressure.

Cullin 3 is a partner of Armadillo repeat containing 5 (ARMC5), the product of the gene responsible for primary bilateral macronodular adrenal hyperplasia

Background ARMC5 has been identified as the gene responsible for PBMAH (primary bilateral macronodular adrenal hyperplasia). ARMC5 inactivating mutations are reported in 20 to 25% of PBMAH patients. ARMC5 is considered as a tumor suppressor gene controlling apoptosis and regulating steroidogenesis. The mechanisms of action of ARMC5 are unknown. The structure of the ARMC5 protein contains ARM repeats and a BTB domain, patterns known to play a role in protein-protein interactions. Therefore identification of proteins that interact with ARMC5 and study of the mechanisms of this interaction will help to understand its function. By co-immunoprecipitation followed by mass spectrometry in HEK293 cells we identified a potential interaction between ARMC5 and Cullin3 (Cul3), also suggested in online databases and by 2 Hybrid Assay (Hu et al., 2017). Cul3 is a protein that mediates the ubiquitination process and subsequent degradation of specific protein substrates. Therefore, the aim of this study was to confirm this interaction and to investigate its mechanisms. Methods We used immunoprecipitation experiments with HA-tagged Cul3 and the bioluminescence resonance energy transfer (BRET) proximity assay in HEK293 cells in order to confirm and investigate the interaction of ARMC5 with Cul3. Results ARMC5 co-immunoprecipitated with HA-Cul3 and a hyperbolic BRET saturation curve was observed with YFP-Cul3 and ARMC5-Luc indicating a specific close proximity between these two proteins. We have also observed that a missense mutation in the BTB domain (p.L754P) of ARMC5 disrupts the interaction with Cul3. Altogether, these complementary approaches demonstrate that ARMC5 and Cul3 form a complex involving the BTB domain of ARMC5. Conclusion These data demonstrate that Cul3 is an ARMC5 partner. A likely direct interaction involves the BTB domain of ARMC5 and can be altered by pathogenic ARMC5 missense mutations. This suggests that ARMC5 participates in the ubiquitination process and open new perspectives in the pathophysiology of PBMAH.

Cullin 3 is a partner of Armadillo Repeat Containing 5 (ARMC5), the product of the gene responsible for Primary Bilateral Macronodular Adrenal Hyperplasia

Background ARMC5 has been identified as the gene responsible for PBMAH (primary bilateral macronodular adrenal hyperplasia). ARMC5 inactivating mutations are reported in 20 to 25% of PBMAH patients. ARMC5 is considered as a tumor suppressor gene controlling apoptosis and regulating steroidogenesis. The mechanisms of action of ARMC5 are unknown. The structure of the ARMC5 protein contains ARM repeats and a BTB domain, patterns known to play a role in protein-protein interactions. Therefore identification of proteins that interact with ARMC5 and study of the mechanisms of this interaction will help to understand its function. By co-immunoprecipitation followed by mass spectrometry in HEK293 cells we identified a potential interaction between ARMC5 and Cullin3 (Cul3), also suggested in online databases and by 2 Hybrid Assay (Hu et al., 2017). Cul3 is a protein that mediates the ubiquitination process and subsequent degradation of specific protein substrates. Therefore, the aim of this study was to confirm this interaction and to investigate its mechanisms. Methods We used immunoprecipitation experiments with HA-tagged Cul3 and the bioluminescence resonance energy transfer (BRET) proximity assay in HEK293 cells in order to confirm and investigate the interaction of ARMC5 with Cul3. Results ARMC5 co-immunoprecipitated with HA-Cul3 and a hyperbolic BRET saturation curve was observed with YFP-Cul3 and ARMC5-Luc indicating a specific close proximity between these two proteins. We have also observed that a missense mutation in the BTB domain (p.L754P) of ARMC5 disrupts the interaction with Cul3. Altogether, these complementary approaches demonstrate that ARMC5 and Cul3 form a complex involving the BTB domain of ARMC5. Conclusion These data demonstrate that Cul3 is an ARMC5 partner. A likely direct interaction involves the BTB domain of ARMC5 and can be altered by pathogenic ARMC5 missense mutations. This suggests that ARMC5 participates in the ubiquitination process and open new perspectives in the pathophysiology of PBMAH.

A novel germline ARMC5 mutation in a patient with bilateral macronodular adrenal hyperplasia: a case report

BackgroundBilateral macronodular adrenal hyperplasia (BMAH) is a rare cause of Cushing’s syndrome (CS). BMAH is predominantly believed to be caused by two mutations, a germline and somatic one, respectively, as described in the two-hit hypothesis. In many familial cases of BMAH, mutations in armadillo repeat containing 5 (ARMC5), a putative tumor suppressor gene, are thought to induce the disorder. The objective of this study was to report a case in which the patient presented with BMAH induced by a novel heterozygous germline ARMC5 mutation (c. 517C > T, p. Arg173*) alone rather than a two-hit mutation.Case presentationA 51-year-old woman was identified with masses in the bilateral adrenals. Serum cortisol levels were increased significantly both in the morning (08:00 AM) and late at night (24:00 AM), while plasma adrenocorticotropic hormone was normal. The patient underwent a left adrenalectomy and histopathology substantiated the BMAH diagnosis. WES of the germline DNA discovered a novel heterozygous germline ARMC5 mutation (c. 517C > T, p. Arg173*) and in silico analysis predicted that the mutation significantly impaired protein function, resulting in inactivated ARMC5. Subsequently, WES of the tumor specimen identified 79 somatic single nucleotide polymorphisms (SNPs)/insertion-deletion (indel) mutations, including 32 missense/nonsense/splice/stop-loss mutations. None of these mutations were CS-related.ConclusionsA novel germline ARMC5 mutation (c. 517C > T, p. Arg173*) was identified that induced BMAH alone without a second mutation. ARMC5 sequencing may improve the identification of clinical forms of BMAH and allow earlier diagnosis of this disease.

ARMC5 mutations in familial and sporadic primary bilateral macronodular adrenal hyperplasia.

To investigate Armadillo repeat-containing 5 (ARMC5) mutations in Chinese patients with familial and sporadic primary bilateral macronodular adrenal hyperplasia (PBMAH), we performed clinical data collection and ARMC5 sequencing for three PBMAH families and 23 sporadic PBMAH patients. ARMC5 pathogenic germline mutations were identified in all 3 PBMAH families. Secondary ARMC5 somatic mutations were found in two adrenal nodules from two PBMAH family members with ARMC5 germline mutations. PBMAH family members with ARMC5 pathogenic germline mutations displayed various clinical manifestations. ARMC5 pathogenic germline mutations were identified in 5 sporadic PBMAH patients among whom one patient displayed both hypercortisolism and primary aldosteronism. We detected a total of 10 ARMC5 pathogenic mutations, of which 8 had not been previously reported. Our results suggest that ARMC5 pathogenic germline mutations are common in familial and sporadic Chinese PBMAH patients, and demonstrate the importance of ARMC5 screening in PBMAH family members to detect patients with insidious PBMAH.

Whole-genome sequencing revealed armadillo repeat containing 5 (ARMC5) mutation in a Chinese family with ACTH-independent macronodular adrenal hyperplasia.

Primary macronodular adrenal hyperplasia (PMAH), also known in the past as bilateral macronodular adrenalhyperplasia or adrenocorticotropin (ACTH)-independent macronodular adrenal hyperplasia, is a rare type of Cushing's syndrome (CS) and is associated with bilateralenlargement of the adrenal glands. It accounts for <1% of all endogenous cases of CS. In order toidentify the pathogenic mutations in the causative gene of (AIMAH pedigrees, Whole-genome sequencing of three patients in family I was used to retrieve candidate causative genes. Meanwhile, the causative gene was identified by Sanger sequencing from the two pedigrees. Sequencing of ARMC5 exons of three patients was carried out to identify somatic mutations. Moreover, haploid clone of one tumor DNA sample was conducted. ARMC5 was the causative gene of two pedigrees confirmed by whole-genome sequencing (WGA) and Sanger sequencing. The variant sites of the two families were c.C943T (p.R315W) and c.C1960T (p.R654X), respectively. Autosomal dominant inheritance of AIMAH was confirmed by genotypes of one family member. Several somatic mutations were discovered in tumor DNA samples. In addition, haploid clone of tumor DNA was confirmed by germline mutation and somaticmutation, which suggested the pathogenic mechanism of "two-hit-model." ARMC5 was the causative gene of AIMAH pedigrees. This AIMAH in this study presented autosomal dominant inheritance, fitting to Mendelian inheritance law. However, the pathogenic mode of this disease showed as compound heterozygote.

ARMC5 is not implicated in familial hyperaldosteronism type II (FH-II)

Germline loss-of-function mutations in the armadillo-repeat-containing 5 (ARMC5) gene are an established cause of Cushing's syndrome due to bilateral macronodular adrenal hyperplasia (BMAH), 1,2 and may play a role in primary aldosteronism. 3 As familial hyperaldosteronism type II (FH-II) has a presumed genetic basis, 4 we hypothesised that germline ARMC5 mutations underlie FH-II. We interrogated whole-exome sequencing data from four FH-II families. We did not identify any pathogenic ARMC5 variants which segregated with the phenotype of primary aldosteronism.

Age-dependent effects of Armc5 haploinsufficiency on adrenocortical function.

Inactivating mutations in the Armadillo repeat-containing 5 (ARMC5) gene have recently been discovered in primary macronodular adrenal hyperplasia (PMAH), a cause of Cushing syndrome. Biallelic ARMC5 inactivation in PMAH suggested that ARMC5 may have tumor suppressor functions in the adrenal cortex. We generated and characterized a new mouse model of Armc5 deficiency. Almost all Armc5 knockout mice died during early embryonic development, around 6.5 and 8.5 days. Knockout embryos did not undergo gastrulation, as demonstrated by the absence of mesoderm development at E7.5. Armc5 heterozygote mice (Armc5+/-) developed normally but at the age of 1 year, their corticosterone levels decreased; this was associated with a decrease of protein kinase A (PKA) catalytic subunit α (Cα) expression both at the RNA and protein levels that were also seen in human patients with PMAH and ARMC5 defects. However, this was transient, as corticosterone levels normalized later, followed by the development of hypercorticosteronemia in one-third of the mice at 18 months of age, which was associated with increases in PKA and Cα expression. Adrenocortical tissue analysis from Armc5+/- mice at 18 months showed an abnormal activation of the Wnt/β-catenin signaling pathway in a subset of zona fasciculata cells. These data confirm that Armc5 plays an important role in early mouse embryonic development. Our new mouse line can be used to study tissue-specific effects of Armc5. Finally, Armc5 haploinsufficiency leads to Cushing syndrome in mice, but only later in life, and this involves PKA, its catalytic subunit Cα, and the Wnt/β-catenin pathway.

ARMC5 mutation in a Portuguese family with primary bilateral macronodular adrenal hyperplasia (PBMAH).

SummaryPBMAH is a rare etiology of Cushing syndrome (CS). Familial clustering suggested a genetic cause that was recently confirmed, after identification of inactivating germline mutations in armadillo repeat-containing 5 (ARMC5) gene. A 70-year-old female patient was admitted due to left femoral neck fracture in May 2014, in Orthopedics Department. During hospitalization, hypertension (HTA) and hypokalemia were diagnosed. She presented with clinical signs of hypercortisolism and was transferred to the Endocrinology ward for suspected CS. Laboratory workup revealed: ACTH <5 pg/mL; urinary free cortisol (UFC), 532 µg/24 h (normal range: 20–90); failure to suppress the low-dose dexamethasone test (0.5 mg every 6 h for 48 h): cortisol 21 µg/dL. Abdominal magnetic resonance imaging (MRI) showed enlarged nodular adrenals (right, 55 × 54 × 30 mm; left, 85 × 53 × 35 mm), and she was submitted to bilateral adrenalectomy. In 2006, this patient’s 39-year-old daughter had been treated by one of the authors. She presented with severe clinical and biological hypercortisolism. Computed tomography (CT) scan showed massively enlarged nodular adrenals with maximal axis of 15 cm for both. Bilateral adrenalectomy was performed. In this familial context of PBMAH, genetic study was performed. Leucocyte DNA genotyping identified in both patients the same germline heterozygous ARMC5 mutation in exon 1 c.172_173insA p.I58Nfs*45. The clinical cases herein described have an identical phenotype with severe hypercortisolism and huge adrenal glands, but different ages at the time of diagnosis. Current knowledge of inheritance of this disease, its insidious nature and the well-known deleterious effect of hypercortisolism favor genetic study to timely identify and treat these patients.Learning points:PBMAH is a rare etiology of CS, characterized by functioning adrenal macronodules and variable cortisol secretion.The asymmetric/asynchronous involvement of only one adrenal gland can also occur, making disease diagnosis a challenge.Familial clustering suggests a genetic cause that was recently confirmed, after identification of inactivating germline mutations in armadillo repeat-containing 5 (ARMC5) gene.The insidious nature of this disease and the well-known deleterious effect of hypercortisolism favor genetic study of other family members, to diagnose and treat these patients timely.As ARMC5 is expressed in many organs and recent findings suggest an association of PBMAH and meningioma, a watchful follow-up is required.