Identification of glucocorticoid-related molecular signature by whole blood methylome analysis

Objective Cushing's syndrome represents a state of excessive glucocorticoids related to glucocorticoid treatments or to endogenous hypercortisolism. Cushing's syndrome is associated with high morbidity, with significant inter-individual variability. Likewise, adrenal insufficiency is a life-threatening condition of cortisol deprivation. Currently, hormone assays contribute to identify Cushing's syndrome or adrenal insufficiency. However, no biomarker directly quantifies the biological glucocorticoid action. The aim of this study was to identify such markers. Design We evaluated whole blood DNA methylome in 94 samples obtained from patients with different glucocorticoid states (Cushing's syndrome, eucortisolism, adrenal insufficiency). We used an independent cohort of 91 samples for validation. Methods Leukocyte DNA was obtained from whole blood samples. Methylome was determined using the Illumina methylation chip array (~850 000 CpG sites). Both unsupervised (principal component analysis) and supervised (Limma) methods were used to explore methylome profiles. A Lasso-penalized regression was used to select optimal discriminating features. Results Whole blood methylation profile was able to discriminate samples by their glucocorticoid status: glucocorticoid excess was associated with DNA hypomethylation, recovering within months after Cushing's syndrome correction. In Cushing's syndrome, an enrichment in hypomethylated CpG sites was observed in the region of FKBP5 gene locus. A methylation predictor of glucocorticoid excess was built on a training cohort and validated on two independent cohorts. Potential CpG sites associated with the risk for specific complications, such as glucocorticoid-related hypertension or osteoporosis, were identified, needing now to be confirmed on independent cohorts. Conclusions Whole blood DNA methylome is dynamically impacted by glucocorticoids. This biomarker could contribute to better assessment of glucocorticoid action beyond hormone assays

Supplementary data from: Steroid profiling using liquid chromatography mass spectrometry during adrenal vein sampling in patients with primary bilateral macronodular adrenocortical hyperplasia (PBMAH)

INTRODUCTION: Adrenal vein sampling (AVS) is not a routine procedure in patients with primary bilateral macronodular adrenocortical hyperplasia (PBMAH), but has been used to determine lateralization of cortisol secretion in order to guide decision of unilateral adrenalectomy. Our aim was to characterize the steroid fingerprints in AVS samples of patients with PBMAH and hypercortisolism and to identify a reference hormone for AVS interpretation. METHOD: Retrospectively, we included 17 patients with PBMAH from the German Cushing’s registry who underwent AVS. 15 steroids were quantified in AVS and peripheral blood samples using LC-MS/MS. We calculated lateralization indices and conversion ratios indicative of steroidogenic enzyme activity to elucidate differences between individual adrenal steroidomes and in steroidogenic pathways. RESULTS: Adrenal volume was negatively correlated with peripheral cortisone (r=0.62, p<0.05). 24-hour urinary free cortisol correlated positively with peripheral androgens (rDHEA=0.57, rDHEAS=0.82, rA=0.73, rT=0.54, p<0.05). DHEA was found to be a powerful reference hormone with high selectivity index, which did not correlate with serume cortisol and has a short half-life. All investigated steroids showed lateralization in single patients indicating the heterogenous steroid secretion pattern in patients with PBMAH. The ratios of corticosterone/aldosterone (catalyzed by CYP11B2), androstenedione/dehydroepiandrosterone (catalyzed by HSD3B2) and cortisone/cortisol (catalyzed by HSD11B2) in adrenal vein samples were higher in smaller adrenals (p<0.05). ARMC5 mutation carriers (n=6) showed lower androstenedione/17-hydroxyprogesterone and higher testosterone/androstenedione (p<0.05) ratios in peripheral blood, in line with lower peripheral androstenedione concentrations (p<0.05). CONCLUSION: Steroid profiling by LC-MS/MS led us to select DHEA as a candidate reference hormone for cortisol secretion. Lateralization and different steroid ratios showed that each steroid and all three steroidogenic pathways may be affected in PBMAH patients. In patients with germline ARMC5 mutations, the androgen pathway was particularly dysregulated

Supplementary data from: Adrenal venous sampling (AVS) in Primary Bilateral Macronodular Adrenocortical Hyperplasia (PBMAH): A study of 16 patients

Adrenal venous sampling in primary bilateral macronodular adrenocortical hyperplasi

Whole blood methylome-derived features to discriminate endocrine hypertension

Background: Arterial hypertension represents a worldwide health burden and a major risk factor for cardiovascular morbidity and mortality. Hypertension can be primary (primary hypertension, PHT), or secondary to endocrine disorders (endocrine hypertension, EHT), such as Cushing's syndrome (CS), primary aldosteronism (PA), and pheochromocytoma/paraganglioma (PPGL). Diagnosis of EHT is currently based on hormone assays. Efficient detection remains challenging, but is crucial to properly orientate patients for diagnostic confirmation and specific treatment. More accurate biomarkers would help in the diagnostic pathway. We hypothesized that each type of endocrine hypertension could be associated with a specific blood DNA methylation signature, which could be used for disease discrimination. To identify such markers, we aimed at exploring the methylome profiles in a cohort of 255 patients with hypertension, either PHT (n = 42) or EHT (n = 213), and at identifying specific discriminating signatures using machine learning approaches. Results: Unsupervised classification of samples showed discrimination of PHT from EHT. CS patients clustered separately from all other patients, whereas PA and PPGL showed an overall overlap. Global methylation was decreased in the CS group compared to PHT. Supervised comparison with PHT identified differentially methylated CpG sites for each type of endocrine hypertension, showing a diffuse genomic location. Among the most differentially methylated genes, FKBP5 was identified in the CS group. Using four different machine learning methods—Lasso (Least Absolute Shrinkage and Selection Operator), Logistic Regression, Random Forest, and Support Vector Machine—predictive models for each type of endocrine hypertension were built on training cohorts (80% of samples for each hypertension type) and estimated on validation cohorts (20% of samples for each hypertension type). Balanced accuracies ranged from 0.55 to 0.74 for predicting EHT, 0.85 to 0.95 for predicting CS, 0.66 to 0.88 for predicting PA, and 0.70 to 0.83 for predicting PPGL. Conclusions: The blood DNA methylome can discriminate endocrine hypertension, with methylation signatures for each type of endocrine disorder

Génétique de l'hyperplasie macronodulaire bilatérale des surrénales : ARMC5 et KDM1A

Context: Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) is a rare disease characterized by the presence of bilateral adrenal macronodules, potentially responsible for a hypercortisolism (Cushing syndrome). A first genetic cause has been identified in 2013: germline inactivating heterozygous mutations of the tumor suppressor gene ARMC5, involved in 20 to 25% of cases. The bilateral adrenal involvement and the description of familial cases without ARMC5 mutation suggest different genetic predisposition factors. PBMAH is a clinically, biologically and radiologically heterogeneous disease with various presentations, from bilateral adrenal incidentalomas without any clinical sign of Cushing syndrome to massively enlarged adrenals associated with severe hypercortisolism. Objectives: To understand the clinical heterogeneity of PBMAH, identify new causing genes, and aim a better clinical, biological and radiological characterization of PBMAH patients. Methods: The clinical data of 352 index patients with ARMC5 genotyping have been collected for the correlation genotype/phenotype. All the known mutations of ARMC5 have been listed, both those already published and those identified in our center. A multi-omics analysis ' with RNAseq, methylome, miRNome, exome sequencing and SNP array ' has been performed in the adrenal tissues from 36 patients with PBMAH treated by adrenalectomy. Results: The analysis of the 352 genotyped index patients confirms that ARMC5 mutated patients have a more pronounced phenotype than wild-type patients, regarding the severity of the Cushing syndrome and the adrenal involvement. 100% of mutated patients had a clear bilateral adrenal involvement on imaging and at least a possible mild autonomous cortisol secretion defined by a plasma cortisol after low dose dexamethasone overnight suppression above 50 nmol/L, while these criteria were inconstant in the non-mutated patients. The association of these two criteria holds a 100% sensitivity for the detection of an ARMC5 mutation. We propose that ARMC5 genotyping should be conditional to the presence of these two simple criteria in order to increase the yield of genetic screening, with a mutation rate near 20%. We present a list of 133 different germline mutations of ARMC5, including 50 never reported in the literature. Most of these mutations are non-sense, missense and frameshift variants. The evaluation of the pathogenic nature of some missense variants may be difficult, and rests on a beam of arguments taking into account the frequency of the variants in population, their segregation in familial cases, in silico predictions, and the functional studies when available. Our functional data allow a better classification for numerous missense variants. The integrative analysis of the genomic data of the samples from the 36 PBMAH patients identifies three distinct molecular groups: G1, comprising the tumors from the 16 ARMC5 mutated patients; G2, which comprises the tumors from the 6 patients with food-dependent Cushing syndrome, mediated by the illegitimate expression of the GIP receptor by adrenocortical cells; and G3, with the tumors from the remaining patients presenting a more heterogeneous phenotype. The analysis of exome data identifies germline inactivating heterozygous mutations of KDM1A in 5/6 G2 patients, constantly associated with a loss of heterozygosity of the short arm of chromosome 1 (1p) harboring the KDM1A locus, thus leading to a biallelic inactivation of the gene. Conclusion: These studies allow a better characterization of PBMAH patients, the identification of predictive criteria for ARMC5 mutation, a better understanding of the clinical and molecular heterogeneity of PBMAH and the discovery of the genetic cause for PBMAH associated with food-dependent Cushing syndrome: the tumor suppressor gene KDM1A.Contexte : L'Hyperplasie Macronodulaire Bilatérale des Surrénales (HMBS) est une maladie rare caractérisée par des macronodules surrénaliens bilatéraux, potentiellement responsables d'un hypercortisolisme (syndrome de Cushing). Une première cause génétique a été identifiée en 2013 : des mutations constitutionnelles inactivatrices hétérozygotes du gène suppresseur de tumeur ARMC5, responsable de 20 à 25% des cas. L'atteinte surrénalienne bilatérale et la description de cas familiaux sans mutation d'ARMC5 suggèrent l'existence d'autres facteurs de prédisposition génétique. L'HMBS est une pathologie hétérogène sur les plans clinique, biologique et radiologique et dont la présentation varie des incidentalomes surrénaliens bilatéraux sans hypercortisolisme clinique à un élargissement massif des surrénales associé à une syndrome de Cushing sévère. Objectifs : Comprendre l'hétérogénéité clinique de l'HMBS, identifier de nouveaux gènes de prédisposition, mieux caractériser les patients sur les plans clinique, biologique et radiologique. Méthodes : Les données cliniques de 352 patients index européens ont été collectées rétrospectivement pour la corrélation génotype/phénotype, l'ensemble des variations d'ARMC5 a été recensé, incluant celles décrites dans la littérature et celles identifiées dans notre centre. Une analyse multi-omique, associant RNAseq, méthylome, miRNome, exome et SNP array, a été réalisée sur les échantillons surrénaliens de 36 patients opérés pour une HMBS. Résultats : L'analyse de la série de 352 patients génotypés pour ARMC5 confirme que les patients mutés ont un phénotype plus marqué que les patients non mutés, en termes de sévérité du syndrome de Cushing et de l'atteinte surrénalienne. 100% des patients mutés ont une atteinte surrénalienne bilatérale claire en imagerie et au moins un hypercortisolisme modéré, défini par un cortisol après freinage par la dexaméthasone supérieur à 50 nmol/L, alors que ces critères sont inconstants chez les patients non mutés. L'association de ces 2 critères a une sensibilité de 100% pour la détection d'une mutation d'ARMC5. Nous proposons que le génotypage d'ARMC5 soit réservé aux patients présentant ces deux critères simples afin d'augmenter le rendement du séquençage, avec un taux de mutation de 20%. Nous présentons 133 mutations constitutionnelles différentes d'ARMC5, dont 50 jamais décrites dans la littérature, représentées majoritairement par des variants non-sens, faux-sens, et des insertions/délétions avec décalage du cadre de lecture. L'évaluation de la pathogénicité des variants faux-sens est parfois difficile, et se base sur leur fréquence en population, leur ségrégation dans les cas familiaux, les prédictions in silico, et les études fonctionnelles disponibles. Nos données fonctionnelles permettent une meilleure classification de nombreux variants faux-sens. L'analyse intégrée des données de génomique des échantillons de 36 patients HMBS révèle trois groupes moléculaires distincts : G1, comprenant les tumeurs des 16 patients porteurs d'une mutation d'ARMC5 ; G2, les tumeurs des 6 patients présentant un syndrome de Cushing dépendant de l'alimentation, médié par l'expression illégitime du récepteur du GIP par les cellules corticosurrénaliennes ; et G3, les tumeurs des autres patients avec phénotype plus hétérogène. L'analyse de l'exome identifie des mutations constitutionnelles inactivatrices hétérozygotes du gène KDM1A chez 5/6 patients du G2, constamment associées à une perte d'hétérozygotie dans la surrénale du bras 1p portant le locus KDM1A, aboutissant à une inactivation biallélique du gène. Conclusion : Ces travaux permettent une meilleure caractérisation des patients porteurs d'HMBS, l'identification de critères prédictifs d'une mutation d'ARMC5, une meilleure compréhension de l'hétérogénéité clinique de l'HMBS et la découverte de la cause génétique de l'HMBS associée à un syndrome de Cushing dépendant de l'alimentation : le gène suppresseur de tumeur KDM1A

Genetics of primary bilateral macronodular adrenal hyperplasia : ARMC5 and KDM1A

Contexte : L'Hyperplasie Macronodulaire Bilatérale des Surrénales (HMBS) est une maladie rare caractérisée par des macronodules surrénaliens bilatéraux, potentiellement responsables d'un hypercortisolisme (syndrome de Cushing). Une première cause génétique a été identifiée en 2013 : des mutations constitutionnelles inactivatrices hétérozygotes du gène suppresseur de tumeur ARMC5, responsable de 20 à 25% des cas. L'atteinte surrénalienne bilatérale et la description de cas familiaux sans mutation d'ARMC5 suggèrent l'existence d'autres facteurs de prédisposition génétique. L'HMBS est une pathologie hétérogène sur les plans clinique, biologique et radiologique et dont la présentation varie des incidentalomes surrénaliens bilatéraux sans hypercortisolisme clinique à un élargissement massif des surrénales associé à une syndrome de Cushing sévère. Objectifs : Comprendre l'hétérogénéité clinique de l'HMBS, identifier de nouveaux gènes de prédisposition, mieux caractériser les patients sur les plans clinique, biologique et radiologique. Méthodes : Les données cliniques de 352 patients index européens ont été collectées rétrospectivement pour la corrélation génotype/phénotype, l'ensemble des variations d'ARMC5 a été recensé, incluant celles décrites dans la littérature et celles identifiées dans notre centre. Une analyse multi-omique, associant RNAseq, méthylome, miRNome, exome et SNP array, a été réalisée sur les échantillons surrénaliens de 36 patients opérés pour une HMBS. Résultats : L'analyse de la série de 352 patients génotypés pour ARMC5 confirme que les patients mutés ont un phénotype plus marqué que les patients non mutés, en termes de sévérité du syndrome de Cushing et de l'atteinte surrénalienne. 100% des patients mutés ont une atteinte surrénalienne bilatérale claire en imagerie et au moins un hypercortisolisme modéré, défini par un cortisol après freinage par la dexaméthasone supérieur à 50 nmol/L, alors que ces critères sont inconstants chez les patients non mutés. L'association de ces 2 critères a une sensibilité de 100% pour la détection d'une mutation d'ARMC5. Nous proposons que le génotypage d'ARMC5 soit réservé aux patients présentant ces deux critères simples afin d'augmenter le rendement du séquençage, avec un taux de mutation de 20%. Nous présentons 133 mutations constitutionnelles différentes d'ARMC5, dont 50 jamais décrites dans la littérature, représentées majoritairement par des variants non-sens, faux-sens, et des insertions/délétions avec décalage du cadre de lecture. L'évaluation de la pathogénicité des variants faux-sens est parfois difficile, et se base sur leur fréquence en population, leur ségrégation dans les cas familiaux, les prédictions in silico, et les études fonctionnelles disponibles. Nos données fonctionnelles permettent une meilleure classification de nombreux variants faux-sens. L'analyse intégrée des données de génomique des échantillons de 36 patients HMBS révèle trois groupes moléculaires distincts : G1, comprenant les tumeurs des 16 patients porteurs d'une mutation d'ARMC5 ; G2, les tumeurs des 6 patients présentant un syndrome de Cushing dépendant de l'alimentation, médié par l'expression illégitime du récepteur du GIP par les cellules corticosurrénaliennes ; et G3, les tumeurs des autres patients avec phénotype plus hétérogène. L'analyse de l'exome identifie des mutations constitutionnelles inactivatrices hétérozygotes du gène KDM1A chez 5/6 patients du G2, constamment associées à une perte d'hétérozygotie dans la surrénale du bras 1p portant le locus KDM1A, aboutissant à une inactivation biallélique du gène. Conclusion : Ces travaux permettent une meilleure caractérisation des patients porteurs d'HMBS, l'identification de critères prédictifs d'une mutation d'ARMC5, une meilleure compréhension de l'hétérogénéité clinique de l'HMBS et la découverte de la cause génétique de l'HMBS associée à un syndrome de Cushing dépendant de l'alimentation : le gène suppresseur de tumeur KDM1A.Context: Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) is a rare disease characterized by the presence of bilateral adrenal macronodules, potentially responsible for a hypercortisolism (Cushing syndrome). A first genetic cause has been identified in 2013: germline inactivating heterozygous mutations of the tumor suppressor gene ARMC5, involved in 20 to 25% of cases. The bilateral adrenal involvement and the description of familial cases without ARMC5 mutation suggest different genetic predisposition factors. PBMAH is a clinically, biologically and radiologically heterogeneous disease with various presentations, from bilateral adrenal incidentalomas without any clinical sign of Cushing syndrome to massively enlarged adrenals associated with severe hypercortisolism. Objectives: To understand the clinical heterogeneity of PBMAH, identify new causing genes, and aim a better clinical, biological and radiological characterization of PBMAH patients. Methods: The clinical data of 352 index patients with ARMC5 genotyping have been collected for the correlation genotype/phenotype. All the known mutations of ARMC5 have been listed, both those already published and those identified in our center. A multi-omics analysis ' with RNAseq, methylome, miRNome, exome sequencing and SNP array ' has been performed in the adrenal tissues from 36 patients with PBMAH treated by adrenalectomy. Results: The analysis of the 352 genotyped index patients confirms that ARMC5 mutated patients have a more pronounced phenotype than wild-type patients, regarding the severity of the Cushing syndrome and the adrenal involvement. 100% of mutated patients had a clear bilateral adrenal involvement on imaging and at least a possible mild autonomous cortisol secretion defined by a plasma cortisol after low dose dexamethasone overnight suppression above 50 nmol/L, while these criteria were inconstant in the non-mutated patients. The association of these two criteria holds a 100% sensitivity for the detection of an ARMC5 mutation. We propose that ARMC5 genotyping should be conditional to the presence of these two simple criteria in order to increase the yield of genetic screening, with a mutation rate near 20%. We present a list of 133 different germline mutations of ARMC5, including 50 never reported in the literature. Most of these mutations are non-sense, missense and frameshift variants. The evaluation of the pathogenic nature of some missense variants may be difficult, and rests on a beam of arguments taking into account the frequency of the variants in population, their segregation in familial cases, in silico predictions, and the functional studies when available. Our functional data allow a better classification for numerous missense variants. The integrative analysis of the genomic data of the samples from the 36 PBMAH patients identifies three distinct molecular groups: G1, comprising the tumors from the 16 ARMC5 mutated patients; G2, which comprises the tumors from the 6 patients with food-dependent Cushing syndrome, mediated by the illegitimate expression of the GIP receptor by adrenocortical cells; and G3, with the tumors from the remaining patients presenting a more heterogeneous phenotype. The analysis of exome data identifies germline inactivating heterozygous mutations of KDM1A in 5/6 G2 patients, constantly associated with a loss of heterozygosity of the short arm of chromosome 1 (1p) harboring the KDM1A locus, thus leading to a biallelic inactivation of the gene. Conclusion: These studies allow a better characterization of PBMAH patients, the identification of predictive criteria for ARMC5 mutation, a better understanding of the clinical and molecular heterogeneity of PBMAH and the discovery of the genetic cause for PBMAH associated with food-dependent Cushing syndrome: the tumor suppressor gene KDM1A

The role of adrenal venous sampling (AVS) in primary bilateral macronodular adrenocortical hyperplasia (PBMAH): a study of 16 patients

OBJECTIVE Primary bilateral macronodular adrenocortical hyperplasia (PBMAH) is a rare cause of ACTH-independent Cushing's syndrome. Current guidelines recommend bilateral adrenalectomy for PBMAH, but several studies showed clinical effectiveness of unilateral adrenalectomy despite bilateral disease in selected patients. Our aim was to evaluate the gain of information which can be obtained through adrenal venous sampling (AVS) based cortisol lateralization ratios for guidance of unilateral adrenalectomy. DESIGN We performed a retrospective analysis of 16 patients with PBMAH and clinical overt cortisol secretion in three centers METHODS: Selectivity of adrenal vein sampling during AVS was defined as a gradient of cortisol or a reference adrenal hormone ≥2.0 between adrenal and peripheral vein. Lateralization was assumed if the dominant to non-dominant ratio of cortisol to reference hormone was ≥4.0. RESULTS AVS was technically successful in all patients based on absolute cortisol levels and in 13 of 16 patients (81%) based on reference hormone levels. Lateralization was documented in 8 of 16 patients. In patients with lateralization, in 5 of 8 cases this occurred toward morphologically larger adrenals, while in 3 patients lateralization was present in bilaterally identical adrenals. The combined volume of adrenals correlated positively with urinary free cortisol, suggesting that adrenal size is the dominant determinant of cortisol secretion. CONCLUSIONS In this study the gain of information through AVS for unilateral adrenalectomy was limited in patients with PBMAH and marked adrenal asymmetry

Identification d’une signature moléculaire d’hypercortisolisme par analyse du methylome du sang total

Part of special issue: 36ème Congrès de la Société Française d’EndocrinologieInternational audienc

Carney complex predisposes to breast cancer: prospective study of 50 women.

International audienceCarney complex (CNC) is a rare genetic syndrome, mostly due to germline loss-of-function pathogenic variants in PRKAR1A. Carney complex includes pigmented skin lesions, cardiac myxomas, primary pigmented nodular adrenocortical dysplasia, and various breast benign tumors