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.

C-peptide level concomitant with hypoglycemia gives better performances than insulin for the diagnosis of endogenous hyperinsulinism: a single-center study of 159 fasting trials.

INTRODUCTION: Diagnosis of endogenous hyperinsulinism relies on the occurrence of a hypoglycemia, concomitant with inadequate high insulin and C-peptide levels. However, diagnostic cutoffs are not consensual among the different learned societies. The objective of this work was to propose optimized cutoffs for these three parameters for the diagnosis of endogenous hyperinsulinism. METHODS: All the patients having performed a fasting trial in Cochin Hospital Endocrinology Department between February 2012 and August 2022 were included. The results of glycemia, insulin and C-peptide levels during fasting trial were collected and analyzed. RESULTS: One hundred and fifty-nine patients were included: 26 with endogenous hyperinsulinism and 133 without endogenous hyperinsulinism. ROC analysis of glycemia nadir during fasting trial identified the value of 2.3 mmol/L as the optimal cutoff, ensuring a sensitivity of 100% associated with a specificity of 81%. ROC analysis of insulin and C-peptide levels concomitant with hypoglycemia <2.3 mmol/L showed very good diagnostic performances of both parameters with respective cutoffs of 3.1 mUI/L (=21.5 pmol/L; sensitivity = 96%; specificity = 92%) and 0.30 nmol/L (sensitivity = 96%; specificity = 100%). Insulin to glycemia ratio as well as C-peptide to glycemia ratio (in pmol/mmol) at the time of glycemia nadir did not show better diagnostic performances than C-peptide alone. CONCLUSION: A C-peptide level 0.3 nmol/L concomitant with a hypoglycemia <2.3 mmol/L appears as the best criterion to make the diagnosis of endogenous hyperinsulinism. Insulin level can be underestimated on hemolyzed blood samples, frequently observed in fasting trial, and thus shows lower diagnostic performances.

Differences in the spectrum of steroidogenic enzyme inhibition between Osilodrostat and Metyrapone in ACTH-dependent Cushing syndrome patients.

Introduction: Osilodrostat is a new 11ß-hydroxylase inhibitor with a mode of action analogous to Metyrapone. The objective of this study was to compare steroidogenic profiles in patients treated with either Osilodrostat or Metyrapone for adrenocorticotrophic hormone (ACTH)-dependent Cushing's syndrome (CS). Methods: Patients followed up at Cochin hospital Endocrinology department between March 2019 and December 2021 for an ACTH-dependent CS, controlled by either Osilodrostat or Metyrapone, were included. A serum profile of five steroids (cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, androstenedione and testosterone) was determined using UPLC- tandem mass spectrometry (UPLC-MS/MS). Results: Nineteen patients treated with Osilodrostat, eight patients treated with Metyrapone and six patients treated with consecutive Metyrapone then Osilodrostat were included. Hypocortisolism (basal cortisol <100 nmol/L) was found in 48% of patients treated with Osilodrostat and 7% of patients treated with Metyrapone. 11-deoxycortisol and androstenedione levels were higher in patients treated with Metyrapone (80.9 (2.2-688.4) and 14.9 (2.5-54.3) nmol/L, respectively) than in patients treated with Osilodrostat (10.3 (0.5-71.9) and 4.0 (0.3-13.3) nmol/L) (P = 0.0009 and P = 0.0005). Testosterone level in women was also higher in Metyrapone group (3.3 (0.93-4.82) nmol/L vs 1.31(0.13-5.09) nmol/L, P = 0.0146). CYP11B1 activity (11-deoxycortisol/cortisol) was not significantly different between the two groups. CYP21A2 activity (17OHprogesterone/11-deoxycortisol) and CYP17A1 activity (17OHprogesterone/androstenedione) were significantly decreased in Osilodrostat group (P < 0.0001). Conclusion: In patients with ACTH-dependent CS, the use of CYP11B1 inhibitors in routine care suggests that Osilodrostat has a less specific effect on the inhibition of steroidogenic enzymes than Metyrapone. This might explain a smaller increase in 11-deoxycortisol and androgen levels in patients treated with Osilodrostat.

Decreased steroidogenic enzyme activity in benign adrenocortical tumors is more pronounced in bilateral lesions as determined by steroid profiling in LC-MS/MS during ACTH stimulation test.

Objective: Large response of steroid precursors, including 17-hydroxyprogesterone, to adrenocorticotropic hormone (ACTH) has been described in adrenocortical tumors, suggesting the existence of intra-tumoral enzymatic deficiencies. This study aimed to compare steroidogenesis enzymes activity in unilateral and bilateral benign tumors using serum steroid profiling in liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in the basal state and after ACTH 1-24 stimulation. Design and methods: A serum profile of seven consecutive adrenal steroids was determined in LC-MS/MS in the basal state (T0) and after ACTH 1-24 stimulation (T60) in 35 patients with bilateral adrenocortical tumors (BL), 38 patients with unilateral tumors (UL) and 37 control subjects (CT). Response amplitude of each individual steroid was evaluated by T60/T0 ratio, whereas enzymatic activity was assessed by the downstream/upstream steroid ratio. Adrenal volume was quantified by a semi-automatic segmentation method. Results: For the seven steroids assayed, the amplitude of response to ACTH was higher in BL than in UL and in CT. The difference between BL and UL persisted even after matching patients on adrenal volume. On glucocorticoids pathway, enzymatic activity of CYP11B1 was significantly decreased in BL (78.3 (43.1-199.4)) in comparison to both UL (122.7 (13.8-228.4), P = 0.0002) and CT (186.8 (42.1-1236.3), P < 0.0001). On mineralocorticoids and androgens pathways, the enzymatic activity of CYP11B2 and CYP17A1-17,20 lyase was also lower in BL than UL and CT. Conclusions: Decreased activity of distal steroidogenesis enzymes CYP11B1, CYP11B2 and CYP17A1-17,20 lyase, responsible for an explosive response to ACTH of upstream precursors in bilateral tumors, limits the synthesis of bioactive steroids, in particular cortisol, despite the increase in adrenal mass. Significance statement: Activity of distal steroidogenesis enzymes (CYP11B1, CYP11B2 and CYP17A1 on glucocorticoids, mineralocorticoids and androgens pathways, respectively) is decreased in adrenocortical benign tumors. This decrease is more pronounced in bilateral lesions and seems to depend more on the nature of the lesion than on the increase in adrenal volume. It is responsible for the explosive response to ACTH of steroid precursors located upstream of these enzymes. It probably allows bioactive steroids, particularly cortisol, to stay in the normal range for a long time despite the increase in adrenal mass.

Identification of predictive criteria for pathogenic variants of primary bilateral macronodular adrenal hyperplasia (PBMAH) gene ARMC5 in 352 unselected patients.

Objective: Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a heterogeneous disease characterized by adrenal macronodules and variable levels of cortisol excess, with not clearly established clinical diagnostic criteria. It can be caused by ARMC5 germline pathogenic variants. In this study, we aimed to identify predictive criteria for ARMC5 variants. Methods: We included 352 consecutive index patients from 12 European centers, sequenced for germline ARMC5 alteration. Clinical, biological and imaging data were collected retrospectively. Results: 52 patients (14.8%) carried ARMC5 germline pathogenic variants and showed a more distinct phenotype than non-mutated patients for cortisol excess (24-h urinary free cortisol 2.32 vs 1.11-fold ULN, respectively, P < 0.001) and adrenal morphology (maximal adrenal diameter 104 vs 83 mm, respectively, P < 0.001) and were more often surgically or medically treated (67.9 vs 36.8%, respectively, P < 0.001). ARMC5-mutated patients showed a constant, bilateral adrenal involvement and at least a possible autonomous cortisol secretion (defined by a plasma cortisol after 1 mg dexamethasone suppression above 50 nmol/L), while these criteria were not systematic in WT patients (78.3%). The association of these two criteria holds a 100% sensitivity and a 100% negative predictive value for ARMC5 pathogenic variant. Conclusion: We report the largest series of index patients investigated for ARMC5 and confirm that ARMC5 pathogenic variants are associated with a more severe phenotype in most cases. To minimize negative ARMC5 screening, genotyping should be limited to clear bilateral adrenal involvement and autonomous cortisol secretion, with an optimum sensitivity for routine clinical practice. These findings will also help to better define PBMAH diagnostic criteria.

Genomic classification of benign adrenocortical lesions.

Benign adrenal tumors cover a spectrum of lesions with distinct morphology and steroid secretion. Current classification is empirical. Beyond a few driver mutations, pathophysiology is not well understood. Here, a pangenomic characterization of benign adrenocortical tumors is proposed, aiming at unbiased classification and new pathophysiological insights. Benign adrenocortical tumors (n = 146) were analyzed by transcriptome, methylome, miRNome, chromosomal alterations and mutational status, using expression arrays, methylation arrays, miRNA sequencing, SNP arrays, and exome or targeted next-generation sequencing respectively. Pathological and hormonal data were collected for all tumors. Pangenomic analysis identifies four distinct molecular categories: (1) tumors responsible for overt Cushing, gathering distinct tumor types, sharing a common cAMP/PKA pathway activation by distinct mechanisms; (2) adenomas with mild autonomous cortisol excess and non-functioning adenomas, associated with beta-catenin mutations; (3) primary macronodular hyperplasia with ARMC5 mutations, showing an ovarian expression signature; (4) aldosterone-producing adrenocortical adenomas, apart from other benign tumors. Epigenetic alterations and steroidogenesis seem associated, including CpG island hypomethylation in tumors with no or mild cortisol secretion, miRNA patterns defining specific molecular groups, and direct regulation of steroidogenic enzyme expression by methylation. Chromosomal alterations and somatic mutations are subclonal, found in less than 2/3 of cells. New pathophysiological insights, including distinct molecular signatures supporting the difference between mild autonomous cortisol excess and overt Cushing, ARMC5 implication into the adreno-gonadal differentiation faith, and the subclonal nature of driver alterations in benign tumors, will orient future research. This first genomic classification provides a large amount of data as a starting point.

Link between steroidogenesis, the cell cycle, and PKA in adrenocortical tumor cells.

Adrenocortical tumors (ACTs) frequently cause steroid excess and present cell-cycle dysregulation. cAMP/PKA signaling is involved in steroid synthesis and play a role in cell-cycle regulation. We investigated, by cell synchronization in the different phases of the cell-cycle, the control of steroidogenesis and the contribution of PKA in adrenocortical cells (H295R and culture of primary pigmented nodular adrenocortical disease cells). Cells showed increased steroidogenesis and a maximal PKA activity at G2 phase, and a reduction at G1 phase. PRKACA overexpression, or cAMP stimulation, enhanced PKA activity and induced steroidogenesis in all synchronized groups but is not sufficient to drive cell-cycle progression. PRKAR1A inactivation enhanced PKA activity and induced STAR gene expression, only in cells in G1, and triggered cell-cycle progression in all groups. These findings provide evidence for a tight association between steroidogenesis and cell-cycle in ACTs. Moreover, PRKAR1A is essential for mediating the function of PKA activity on both steroidogenesis and cell-cycle progression in adrenocortical cells.

Cullin 3 targets the tumor suppressor gene ARMC5 for ubiquitination and degradation.

ARMC5 (Armadillo repeat containing 5 gene) was identified as a new tumor suppressor gene responsible for hereditary adrenocortical tumors and meningiomas. ARMC5 is ubiquitously expressed and encodes a protein which contains a N-terminal Armadillo repeat domain and a C-terminal BTB (Bric-a-Brac, Tramtrack and Broad-complex) domain, both docking platforms for numerous proteins. At present, expression regulation and mechanisms of action of ARMC5 are almost unknown. In this study, we showed that ARMC5 interacts with CUL3 requiring its BTB domain. This interaction leads to ARMC5 ubiquitination and further degradation by the proteasome. ARMC5 alters cell cycle (G1/S phases and cyclin E accumulation) and this effect is blocked by CUL3. Moreover, missense mutants in the BTB domain of ARMC5, identified in patients with multiple adrenocortical tumors, are neither able to interact and be degraded by CUL3/proteasome nor alter cell cycle. These data show a new mechanism of regulation of the ARMC5 protein and open new perspectives in the understanding of its tumor suppressor activity.

Mass spectrometry-based steroid profiling in primary bilateral macronodular adrenocortical hyperplasia.

Biochemical characterization of primary bilateral macronodular adrenocortical hyperplasia (PBMAH) by distinct plasma steroid profiles and its putative correlation to disease has not been previously studied. LC-MS/MS-based steroid profiling of 16 plasma steroids was applied to 36 subjects (22 females, 14 males) with PBMAH, 19 subjects (16 females, 3 males) with other forms of adrenal Cushing's syndrome (ACS), and an age and sex-matched control group. Germline ARMC5 sequencing was performed in all PBMAH cases. Compared to controls, PBMAH showed increased plasma 11-deoxycortisol, corticosterone, 11-deoxycorticosterone, 18-hydroxycortisol, and aldosterone, but lower progesterone, DHEA, and DHEA-S with distinct differences in subjects with and without pathogenic variants in ARMC5. Steroids that showed isolated differences included cortisol and 18-oxocortisol with higher (P < 0.05) concentrations in ACS than in controls and aldosterone with higher concentrations in PBMAH when compared to controls. Larger differences in PBMAH than with ACS were most clear for corticosterone, but there were also trends in this direction for 18-hydroxycortisol and aldosterone. Logistic regression analysis indicated four steroids - DHEA, 11-deoxycortisol, 18-oxocortisol, and corticosterone - with the most power for distinguishing the groups. Discriminant analyses with step-wise variable selection indicated correct classification of 95.2% of all subjects of the four groups using a panel of nine steroids; correct classification of subjects with and without germline variants in ARMC5 was achieved in 91.7% of subjects with PBMAH. Subjects with PBMAH show distinctive plasma steroid profiles that may offer a supplementary single-test alternative for screening purposes.