Functional Characterization of PRKAR1A Mutations Reveals a Unique Molecular Mechanism Causing Acrodysostosis but Multiple Mechanisms Causing Carney Complex.

The main target of cAMP is PKA, the main regulatory subunit of which (PRKAR1A) presents mutations in two genetic disorders: acrodysostosis and Carney complex. In addition to the initial recurrent mutation (R368X) of the PRKAR1A gene, several missense and nonsense mutations have been observed recently in acrodysostosis with hormonal resistance. These mutations are located in one of the two cAMP-binding domains of the protein, and their functional characterization is presented here. Expression of each of the PRKAR1A mutants results in a reduction of forskolin-induced PKA activation (measured by a reporter assay) and an impaired ability of cAMP to dissociate PRKAR1A from the catalytic PKA subunits by BRET assay. Modeling studies and sensitivity to cAMP analogs specific for domain A (8-piperidinoadenosine 3',5'-cyclic monophosphate) or domain B (8-(6-aminohexyl)aminoadenosine-3',5'-cyclic monophosphate) indicate that the mutations impair cAMP binding locally in the domain containing the mutation. Interestingly, two of these mutations affect amino acids for which alternative amino acid substitutions have been reported to cause the Carney complex phenotype. To decipher the molecular mechanism through which homologous substitutions can produce such strikingly different clinical phenotypes, we studied these mutations using the same approaches. Interestingly, the Carney mutants also demonstrated resistance to cAMP, but they expressed additional functional defects, including accelerated PRKAR1A protein degradation. These data demonstrate that a cAMP binding defect is the common molecular mechanism for resistance of PKA activation in acrodysosotosis and that several distinct mechanisms lead to constitutive PKA activation in Carney complex.

Urine steroid metabolomics as a diagnostic tool in primary aldosteronism.

Primary aldosteronism (PA) causes 5-10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and glucocorticoid precursors. Urine steroid metabolomics identified patients with PA with high accuracy, both when applied to all 34 or only the three most discriminative steroid metabolites (average areas under the receiver-operating characteristics curve [AUCs-ROC] 0.95-0.97). Whilst machine learning was suboptimal in differentiating UPA from BPA (average AUCs-ROC 0.65-0.73), it readily identified APA cases harbouring somatic KCNJ5 mutations (average AUCs-ROC 0.79-85). These patients showed a distinctly increased urine excretion of the hybrid steroid 18-hydroxycortisol and its metabolite 18-oxo-tetrahydrocortisol, the latter identified by machine learning as by far the most discriminative steroid. In conclusion, urine steroid metabolomics is a non-invasive candidate test for the accurate identification of PA cases and KCNJ5-mutated APAs.

Histological Characterization of Aldosterone-producing Adrenocortical Adenomas with Different Somatic Mutations.

CONTEXT: Aldosterone-producing adrenocortical adenomas (APAs) are mainly composed of clear (lipid rich) and compact (eosinophilic) tumor cells. The detailed association between these histological features and somatic mutations (KCNJ5, ATP1A1, ATP2B3, and CACNA1D) in APAs is unknown. OBJECTIVE: To examine the association between histological features and individual genotypes in APAs. METHODS: Examination of 39 APAs subjected to targeted next-generation sequencing (11 KCNJ5, 10 ATP1A1, 10 ATP2B3, and 8 CACNA1D) and quantitative morphological and immunohistochemical (CYP11B2 and CYP17A1) analyses using digital imaging software. RESULTS: KCNJ5- and ATP2B3-mutated APAs had clear cell dominant features (KCNJ5: clear 59.8% [54.4-64.6%] vs compact 40.2% (35.4-45.6%), P = .0022; ATP2B3: clear 54.3% [48.2-62.4 %] vs compact 45.7% (37.6-51.8 %), P = .0696). ATP1A1- and CACNA1D-mutated APAs presented with marked intratumoral heterogeneity. A significantly positive correlation of immunoreactivity was detected between CYP11B2 and CYP17A1 in tumor cells of KCNJ5-mutated APAs (P = .0112; ρ = 0.7237), in contrast, significantly inverse correlation was detected in ATP1A1-mutated APAs (P = .0025; ρ = -0.8667). CONCLUSION: KCNJ5-mutated APAs, coexpressing CYP11B2 and CYP17A1, were more deviated in terms of zonation-specific differentiation of adrenocortical cells than ATP1A1- and ATP2B3-mutated APAs.

In situ metabolomics of aldosterone-producing adenomas.

Recent genetic examinations and multisteroid profiles have provided the basis for subclassification of aldosterone-producing adenomas (APAs). The objective of the current study was to produce a comprehensive, high-resolution mass spectrometry imaging (MSI) map of APAs in relation to morphometry, immunohistochemical profiles, mutational status, and clinical outcome. The study cohort comprised 136 patients with unilateral primary aldosteronism. Matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance MSI was conducted, and metabolite profiles were analyzed with genotype/phenotype information, including digital image analysis from morphometry and IHC of steroidogenic enzymes. Distinct molecular signatures between KCNJ5- and CACNA1D-mutated APAs with significant differences of 137 metabolites, including metabolites of purine metabolism and steroidogenesis, were observed. Intratumor concentration of 18-oxocortisol and 18-hydroxycortisol were inversely correlated with the staining intensity of CYP11B1. Lower staining intensity of CYP11B1 and higher levels of 18-oxocortisol were associated with a higher probability of complete clinical success after surgery. The present study demonstrates distinct metabolomic profiles of APAs in relation to tumor genotype. In addition, we reveal an inverse correlation between cortisol derivatives and CYP11B1 and the impact of 18-oxocortisol and CYP11B1 on clinical outcome, which provides unprecedented insights into the pathophysiology, clinical features, and steroidogenesis of APAs.

Chain-breaking activity of resveratrol and piceatannol in a linoleate micellar model.

This study investigated the in vitro protective effects of three derivatives of resveratrol, i.e., piceatannol (trans-3,5,3',4'-tetrahydroxystilbene), PDM2 (1,3-dichloro-5-[(1E)-2-(4-chlorophenyl)ethenyl]-benzene) and PDM11 ((E)-5-[2-(4-chlorophenyl)ethenyl]-1,3-dimethoxyphenyl-ethene), compared with resveratrol as reference compound, against oxidation of linoleate micelles (10(-2)M) initiated by radiolysis-generated hydroxyl radicals. Lipid peroxidation was monitored by conjugated dienes (differential absorbance at 234nm), and by hydroperoxides (reverse phase HPLC with chemiluminescence detection). The higher the concentration of resveratrol or piceatannol (from 10(-5)M to 10(-4)M), the stronger the antioxidant ability. Piceatannol, with the presence of an additional hydroxyl group, showed a better antioxidant effect than resveratrol for a given concentration (competition with the fatty acid to scavenge lipid peroxyl radicals LOO), whereas PDM2 and PDM11, without any hydroxyl group, did not exhibit any significant protective effect. A lower limit for the LOO rate constant has been estimated for piceatannol (>/=1.4x10(5)M(-1)s(-1)) and for resveratrol (>/=0.3x10(5)M(-1)s(-1)).

Targeting CXCR4 (CXC Chemokine Receptor Type 4) for Molecular Imaging of Aldosterone-Producing Adenoma.

Primary aldosteronism is the most frequent cause of secondary hypertension and is associated with increased morbidity and mortality compared with hypertensive controls. The central diagnostic challenge is the differentiation between bilateral and unilateral disease, which determines treatment options. Bilateral adrenal venous sampling, currently recommended for differential diagnosis, is an invasive procedure with several drawbacks, making it desirable to develop novel noninvasive diagnostic tools. When investigating the expression pattern of chemokine receptors by quantitative real-time polymerase chain reaction and immunohistochemistry, we observed high expression of CXCR4 (CXC chemokine receptor type 4) in aldosterone-producing tissue in normal adrenals, adjacent adrenal cortex from adrenocortical adenomas, and in aldosterone-producing adenomas (APA), correlating strongly with the expression of CYP11B2 (aldosterone synthase). In contrast, CXCR4 was not detected in the majority of nonfunctioning adenomas that are frequently found coincidently. The specific CXCR4 ligand 68Ga-pentixafor has recently been established as radiotracer for molecular imaging of CXCR4 expression and showed strong and specific binding to cryosections of APAs in our study. We further investigated 9 patients with primary aldosteronism because of APA by 68Ga-pentixafor-positron emission tomography. The tracer uptake was significantly higher on the side of increased adrenocortical aldosterone secretion in patients with APAs compared with patients investigated by 68Ga-pentixafor-positron emission tomography for other causes. Molecular imaging of aldosterone-producing tissue by a CXCR4-specific ligand may, therefore, be a highly promising tool for noninvasive characterization of patients with APAs.

Histopathological and genetic characterization of aldosterone-producing adenomas with concurrent subclinical cortisol hypersecretion: a case series.

PURPOSE: Aldosterone-producing adenomas with concurrent subclinical cortisol hypersecretion are reported in an increasing number of patients. Five aldosterone-producing adenomas from patients with primary aldosteronism and subclinical hypercortisolism were examined. THE AIMS OF OUR STUDY WERE: (1) to analyze pathological features and immunohistochemical expression of CYP11B1 (11ß-hydroxylase) and CYP11B2 (aldosterone synthase) in these tumors; (2) to investigate somatic mutations involved in adrenal steroid hypersecretion and/or tumor growth. METHODS: Archival micro-dissected paraffin-embedded slides from tumor specimens were used for histological and molecular studies. Immunohistochemistry was performed using monoclonal anti-CYP11B1 and anti-CYP11B2 antibodies. Cellular composition was determined by examining for known features of zona fasciculata and zona glomerulosa, and immunoreactivity for CYP11B1 and CYP11B2 by McCarty H-score. Spot regions for mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D, PRKACA, and CTNNB1 gene sequences were evaluated. RESULTS: Four APAs showed a predominant (≥50%) zona fasciculata-like cell pattern: one tumor had CYP11B1 H-score = 150, no detectable CYP11B2 expression, and harbored a PRKACA p.Leu206Arg mutation (that we have reported previously elsewhere), one had no CYP11B1 expression, CYP11B2 H-score = 40, and no mutations; the remaining two adenomas had high CYP11B1 H-score (160 and 240, respectively) and low CYP11B2 H-score (30 and 15, respectively), with the latter harboring a CTNNB1 p.Ser45Phe activating mutation. One of five aldosterone-producing adenomas had a predominant zona glomerulosa-like pattern, CYP11B1 H-score = 15, CYP11B2 H-score = 180, and no mutations. CONCLUSIONS: The majority of aldosterone-producing adenomas with concurrent subclinical cortisol hypersecretion were composed mainly of zona fasciculata-like cells, while CYP11B1 and CYP11B2 immunostaining demonstrated clear heterogeneity. In a subset of cases, different somatic mutations may be involved in hormone excess and tumor formation.

Steroid metabolome analysis reveals prevalent glucocorticoid excess in primary aldosteronism.

BACKGROUND: Adrenal aldosterone excess is the most common cause of secondary hypertension and is associated with increased cardiovascular morbidity. However, adverse metabolic risk in primary aldosteronism extends beyond hypertension, with increased rates of insulin resistance, type 2 diabetes, and osteoporosis, which cannot be easily explained by aldosterone excess. METHODS: We performed mass spectrometry-based analysis of a 24-hour urine steroid metabolome in 174 newly diagnosed patients with primary aldosteronism (103 unilateral adenomas, 71 bilateral adrenal hyperplasias) in comparison to 162 healthy controls, 56 patients with endocrine inactive adrenal adenoma, 104 patients with mild subclinical, and 47 with clinically overt adrenal cortisol excess. We also analyzed the expression of cortisol-producing CYP11B1 and aldosterone-producing CYP11B2 enzymes in adenoma tissue from 57 patients with aldosterone-producing adenoma, employing immunohistochemistry with digital image analysis. RESULTS: Primary aldosteronism patients had significantly increased cortisol and total glucocorticoid metabolite excretion (all P < 0.001), only exceeded by glucocorticoid output in patients with clinically overt adrenal Cushing syndrome. Several surrogate parameters of metabolic risk correlated significantly with glucocorticoid but not mineralocorticoid output. Intratumoral CYP11B1 expression was significantly associated with the corresponding in vivo glucocorticoid excretion. Unilateral adrenalectomy resolved both mineralocorticoid and glucocorticoid excess. Postoperative evidence of adrenal insufficiency was found in 13 (29%) of 45 consecutively tested patients. CONCLUSION: Our data indicate that glucocorticoid cosecretion is frequently found in primary aldosteronism and contributes to associated metabolic risk. Mineralocorticoid receptor antagonist therapy alone may not be sufficient to counteract adverse metabolic risk in medically treated patients with primary aldosteronism. FUNDING: Medical Research Council UK, Wellcome Trust, European Commission.

PRKACA Somatic Mutations Are Rare Findings in Aldosterone-Producing Adenomas.

CONTEXT: Somatic mutations have been found causative for endocrine autonomy in aldosterone-producing adenomas (APAs). Whereas mutations of PRKACA (catalytic subunit of protein kinase A) have been identified in cortisol-producing adenomas, the presence of PRKACA variants in APAs is unknown, especially in those that display cosecretion of cortisol. OBJECTIVE: The objective of the study was to investigate PRKACA somatic variants identified in APA cases. DESIGN: Identification of PRKACA somatic variants in APAs by whole-exome sequencing followed by in vitro analysis of the enzymatic activity of PRKACA variants and functional characterization by double immunofluorescence of CYP11B2 and CYP11B1 expression in the corresponding tumor tissues. SETTING AND PATIENTS: APA tissues were collected from 122 patients who underwent unilateral adrenalectomy for primary aldosteronism between 2005 and 2015 at a single institution. RESULTS: PRKACA somatic mutations were identified in two APA cases (1.6%). One APA carried a newly identified p.His88Asp variant, whereas in a second case, a p.Leu206Arg mutation was found, previously described only in cortisol-producing adenomas with overt Cushing's syndrome. Functional analysis showed that the p.His88Asp variant was not associated with gain of function. Although CYP11B2 was strongly expressed in the p.His88Asp-mutated APA, the p.Leu206Arg carrying APA predominantly expressed CYP11B1. Accordingly, biochemical Cushing's syndrome was present only in the patient with the p.Leu206Arg mutation. After adrenalectomy, both patients improved with a reduced number of antihypertensive medications and normalized serum potassium levels. CONCLUSIONS: We describe for the first time PRKACA mutations as rare findings associated with unilateral primary aldosteronism. As cortisol cosecretion occurs in a subgroup of APAs, other molecular mechanisms are likely to exist.

Genotype-Specific Steroid Profiles Associated With Aldosterone-Producing Adenomas.

Primary aldosteronism comprises 2 main subtypes: unilateral aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia. Somatic KCNJ5 mutations are found in APA at a prevalence of around 40% that drive and sustain aldosterone excess. Somatic APA mutations have been described in other genes (CACNA1D, ATP1A1, and ATP2B3) albeit at a lower frequency. Our objective was to identify genotype-specific steroid profiles in adrenal venous (AV) and peripheral venous (PV) plasma in patients with APAs. We measured the concentrations of 15 steroids in AV and PV plasma samples by liquid chromatography-tandem mass spectrometry from 79 patients with confirmed unilateral primary aldosteronism. AV sampling lateralization ratios of steroids normalized either to cortisol or to DHEA+androstenedione were also calculated. The hybrid steroid 18-oxocortisol exhibited 18- and 16-fold higher concentrations in lateralized AV and PV plasma, respectively, from APA with KCNJ5 mutations compared with all other APA combined together (P<0.001). Lateralization ratios for the KCNJ5 group were also generally higher. Strikingly, we demonstrate that a distinct steroid signature can differentiate APA genotype in AV and PV plasma. Notably, a 7-steroid fingerprint in PV plasma correctly classified 92% of the APA according to genotype. Prospective studies are necessary to translate these findings into clinical practice and determine if steroid fingerprinting could be of value to select patients with primary aldosteronism who are particularly suitable candidates for adrenal venous sampling because of a high probability of having an APA.

Duration of postoperative fibrinolysis after total hip or knee replacement: a laboratory follow-up study.

INTRODUCTION: Hyperfibrinolysis is observed during and immediately after major orthopedic surgery. The kinetics and duration of this phase should be defined to adjust the duration of antifibrinolytic treatment with tranexamic acid (TXA). OBJECTIVE: We aimed to quantify the duration of postoperative fibrinolysis and to assess the biological impact of TXA administration. MATERIALS AND METHODS: Fourteen patients undergoing total hip replacement (THR) and 10 patients undergoing total knee replacement (TKR) with tourniquet were included in an observational, prospective, single-center study. Among these patients, 7 THR patients and 5 TKR patients received TXA (15mg/kg IV intraoperatively, followed by continuous infusion of 15mg/kg/h until end of surgery, then every 4hours until 16±2hours after surgery). D-dimers, euglobulin lysis time (ELT), and thrombin generation time (TGT) were measured prior to surgery as well as 6, 18 and 24hours (H) after. RESULTS: No significant difference in ELT was observed between the groups. In contrast, D-dimers significantly increased postoperatively in patients not treated with TXA (p<0.001), while such an increase was prevented in patients receiving TXA, as measured at H0, H6, H18 and H24 after THR, and at H6 and H18 after TKR (p<0.001). No significant between-group change in TGT, was observed (peak thrombin and endogenous thrombin potential) all along the study. CONCLUSION: This study shows that fibrinolysis peaked 6hours after end of surgery and maintained about 18hours after surgery, as evidenced by an increase in D-dimers. When administered for up to 16±2hours after surgery, TXA reduced postoperative fibrinolysis.

Simple spectrophotometric assessment of the trans-/cis-resveratrol ratio in aqueous solutions.

The solubility and molar absorptivity of trans- and cis-resveratrol isomers in aqueous solvents are poorly described. This study aimed to develop and describe a new simple method for the determination of trans- and cis-resveratrol concentrations in aqueous solutions. Up to 300 microM trans-resveratrol was dissolved in water by sonication for 2h. Cis-resveratrol was obtained by exposing a 100-muM trans-resveratrol aqueous solution to sunlight for 8h, followed by HPLC separation and analysis by mass spectrometry (resveratrol oxidation products were absent). Accurate values for UV absorbance in water were [see text], epsilon(286 nm)=23400 M(-1)cm(-1) for trans-resveratrol and [see text], epsilon(304nm)=9515 M(-1)cm(-1) for cis-resveratrol. These values allowed us to propose formulae to assess the trans-/cis-resveratrol ratio in water, using a simple and reliable UV-vis spectrophotometric method. Statistical analysis revealed no significant difference between our UV method and the commonly used HPLC method. All these data are transferable to 150 mM NaCl and 10 mM phosphate buffer solutions, which could be particularly useful for cell culture, ex vivo and in vivo studies.