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During follicular development, a few dominant follicles develop to large antral dominant follicles, whereas the remaining follicles undergo atretic degeneration. Because vascularization on the follicular surface is a morphological feature of dominant follicles, we previously classified these follicles as vascularized follicles (VFs) and non-VFs (NVFs). In NVFs, progesterone producing genes were expressed similarly to that in VFs; however, the progesterone concentration in follicular fluid was low in large NVFs. Therefore, we estimated that progesterone is converted to cortisol, which induces the loss of follicular functions. In this study, we comparative analyzed the expression of genes for progesterone converting enzymes (Cytochrome (CYP)11B1, CYP21A2, Hydroxysteroid (HSD)11B2) and cortisol receptor (NR3C1) in VF and NVF granulosa cells. In NVFs, expression of cortisol producing genes (CYP11B1 and CYP21A2) was higher than in VFs. Expression of the gene for the cortisol metabolizing enzyme HSD11B2 in NVFs was significantly lower than in VFs. In NVFs, accompanied by increasing cortisol concentration in follicular fluid, apoptosis of granulosa and cumulus cells was observed. Cultivation with FSH and metyrapone (a CYP11B1 inhibitor) of NVF cumulus–oocyte complexes inhibited apoptosis of cumulus cells and induced cumulus cell proliferation and oocyte maturation. Cortisol-induced CYP11B1 and CYP21A2 expression, whereas FSH-induced HSD11B2 mRNA expression in VF granulosa cells in the presence of cortisol. Furthermore, an addition of 18β-glycyrrhetinic acid (18-GA; a HSD17B2 inhibitor) to cortisol and FSH-containing medium increased apoptosis of VF granulosa cells. These results suggested that cortisol is a stimulatory factor that induces follicular atresia; furthermore, inhibition of cortisol production by FSH might increase the number of healthy preovulatory follicles in pigs.

Primary aldosteronism (PA) is a common cause of secondary hypertension. Recent technological advances in genetic analysis have provided a better understanding of the molecular pathogenesis of this disease. The application of next-generation sequencing has resulted in the identification of somatic mutations in aldosterone-producing adenoma (APA), a major subtype of PA. Based on the recent findings using a sequencing method that selectively targets the tumor region where aldosterone synthase (CYP11B2) is expressed, the vast majority of APAs appear to harbor a somatic mutation in one of the aldosterone-driver genes, including KCNJ5, ATP1A1, ATP2B3, CACNA1D, CACNA1H, and CLCN2. Mutations in these genes alter intracellular ion homeostasis and enhance aldosterone production. In a small subset of APAs, somatic activating mutations in the CTNNB1 gene, which encodes β-catenin, have also been detected. Accumulating evidence suggests that race and sex impact the somatic mutation spectrum of APA. Specifically, somatic mutations in the KCNJ5 gene, encoding an inwardly rectifying K+ channel, are common in APAs from Asian populations as well as women regardless of race. Associations between APA histology, genotype, and patient clinical characteristics have also been proposed, suggesting a potential need to consider race and sex for the management of PA patients. Herein, we review recent findings regarding somatic mutations in APA and discuss potential roles of race and sex on the pathophysiology of APA as well as possible clinical implications.

Background Glucocorticoid-remediable aldosteronism (GRA) is a form of heritable hypertension caused by a chimeric fusion resulting from unequal crossing over between 11β‐hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2), which are two genes with similar sequences. Different crossover patterns of the CYP11B1 and CYP11B2 chimeric genes may be associated with a variety of clinical presentations. It is therefore necessary to develop an efficient approach for identifying the differences between the hybrid genes of a patient with GRA. Results We developed a long-read analysis pipeline named GRAde (GRA deciphering), which utilizes the nonidentical bases in the CYP11B1 and CYP11B2 genomic sequences to identify and visualize the chimeric form. We sequenced the polymerase chain reaction (PCR) products of the CYP11B1/CYP11B2 chimeric gene from 36 patients with GRA using the Nanopore MinION device and analyzed the sequences using GRAde. Crossover events were identified for 30 out of the 36 samples. The crossover sites appeared in the region exhibiting high sequence similarity between CYP11B1 and CYP11B2, and 53.3% of the cases were identified as having a gene conversion in intron 2. More importantly, there were six cases for whom the PCR products indicated a chimeric gene, but the GRAde results revealed no crossover pattern. The crossover regions were further verified by Sanger sequencing analysis. Conclusions PCR-based target enrichment followed by long-read sequencing is an efficient and precise approach to dissecting complex genomic regions, such as those involved in GRA mutations, which could be directly applied to clinical diagnosis. The scripts of GRAde are available at https://github.com/hsu-binfo/GRAde.

Emerging evidence supports that exposure to ambient fine particulate matter (PM2.5) is associated with the metabolic syndrome. As the main neuroendocrine axis in mammals, the hypothalamic-pituitary-adrenal (HPA) axis's circadian rhythm (CR) plays an essential role in regulating metabolic homeostasis. Our previous studies found that ambient PM2.5 exposure caused CR disorder of the critical enzymes involved in lipid metabolism in mouse liver and adipose tissues. However, the impact of ambient PM2.5 exposure on the HPA axis is not fully illustrated yet. Male C57BL/6 mice were randomly exposed to ambient PM2.5 or filtered air for ten weeks via a whole-body exposure system. Rhythmic oscillations of clock genes in the hypothalamus and adrenal gland were characterized. The effects of ambient PM2.5 exposure on clock gene expression and rhythmic expression of molecules related to glucocorticoid synthesis were also examined. Firstly, a more robust CR of clock genes was demonstrated in the adrenal gland than that in the hypothalamus. Secondly, PM2.5 exposure significantly inhibited the expression of Clock at ZT8 in the hypothalamus. However, both circadian oscillation and expression levels of Bmal1, Cry1, Cry2, and Rorα were increased significantly by ambient PM2.5 exposure in the adrenal gland. Moreover, abnormal rhythmic oscillation patterns of corticotropin-releasing hormone and adrenocorticotropic hormone were observed after ambient PM2.5 exposure, with no change at the expression levels. Finally, the expression of Cyp11b1 was markedly decreased at ZT0 in the adrenal gland of PM2.5 exposed mice. Our findings provide new insights into the ambient PM2.5 exposure-induced metabolic syndrome from the perspective of CR disturbances.

11β-hydroxylase deficiency is a rare autosomal recessive disorder due to impaired steroidogenesis in the adrenal cortex caused by pathogenic mutations in the CYP11B1 gene. The main clinical manifestations are determined by a deficiency of cortisol, ACTH hyperproduction, excessive androgens secretion and the accumulation of 11-deoxycorticosterone, which leads to the development of arterial hypertension. In the diagnostic search, it is important to take into account the ethnicity of the patient, since the frequency of the disease and the prevalence of mutations differ between ethnic groups. The article presents a clinical case of 11β-hydroxylase deficiency as the result of compound heterozygous mutations in the CYP11B1 gene in a patient of Turkic origin. This case shows the clinical manifestations and the development of complications of 11β-hydroxylase deficiency, the stages of differential diagnosis of patients with 21-hydroxylase deficiency.

Hydroxylation of steroids has acquired special relevance for the pharmaceutical industries. Particularly, the 11β-hydroxylation of steroids is a reaction of biotechnological importance currently carried out at industrial scale by the fungus Cochliobolus lunatus. In this work, we have identified the genes encoding the cytochrome CYP103168 and the reductase CPR64795 of C. lunatus responsible for the 11β-hydroxylase activity in this fungus, which is the key step for the preparative synthesis of cortisol in industry. A recombinant Corynebacterium glutamicum strain harbouring a plasmid expressing both genes forming a synthetic bacterial operon was able to 11β-hydroxylate several steroids as substrates. This is a new example to show that the industrial strain C. glutamicum can be used as a suitable chassis to perform steroid biotransformation expressing eukaryotic cytochromes., This work was supported by grants from the Ministry of Science and Innovation (BFU2006-15214-C03-01, BFU2009-11545-C03-03) and Ministry of Economy and Competitiveness (BIO2012-39695-C02-01).

The genetic factors related to early-onset hypertension are largely unknown. This study aimed to determine the spectrum of steroid metabolism gene variants and the clinical relationships of these variants to phenotypes in Chinese patients with early-onset hypertension. A total of 306 consecutive early-onset hypertensive patients were recruited. All coding exons and flanking intronic regions of KCNJ5, CYP11B1, and CYP17A1 were sequenced. Long-distance polymerase chain reaction was used to search for a CYP11B1/CYP11B2 chimeric gene. Pedigree investigations and genotype-phenotype analyses were performed for patients with rare variants. Nine rare variants were detected in eight patients (2.6%), but no CYP11B1/CYP11B2 chimeric gene was identified. One patient and two of her siblings were found to carry compound heterozygous mutations (C183Y and T390R) in CYP17A1 and were eventually diagnosed with atypical congenital adrenal hyperplasia. Patients with rare variants had younger ages of onset [17 (16, 20) vs. 30 (23, 35) years old, p = 0.010] and higher systolic blood pressure (148.5 ± 9.6 vs. 137.9 ± 17.8 mmHg, p = 0.021) than those without rare variants. Additionally, the patients and their relatives carrying rare variants exhibited increased serum free corticosterone [230.4 (7.4, 533.0) vs. 1.9 (0.9, 6.7)ng/ml, p = 0.001] and 11-deoxycorticosterone [16.16 (0.59, 33.23) vs. 0.77 (0.41, 0.96)ng/ml, p = 0.038] levels. Genetic testing is useful for the etiologic diagnosis of early-onset hypertension. Rare variants in steroid metabolism genes were associated with more severe clinical expression and abnormal circulating steroid metabolites in patients with early-onset hypertension.

To assess the diagnostic performance of the H-score of 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the histopathological diagnosis of adrenocortical tumors (ACT). We retrospectively evaluated 199 cases of ACT, of which 85 were diagnosed as aldosterone-producing adenoma (APA), 66 as cortisol-producing adenoma (CPA), 9 as aldosterone–cortisol co-secreting adenoma, 30 as nonhyperfunctioning adenoma, and 9 as adrenocortical carcinoma (ACC). Immunohistochemical staining was performed using anti-CYP11B1 and anti-CYP11B2 monoclonal antibodies. The staining was quantified by the McCarty’s H-score system. The diagnostic performance was assessed by the receiver operating characteristic curve (ROC). The H-score of CYP11B1 is highest in the CPA group and lowest in the ACC group. The H-score of CYP11B2 in the APA group is significantly higher than other ACT groups. The area under ROC (AUC) of an increased H-score of CYP11B2 (>65) for the diagnosis of APA was 0.971 (95%CI 0.937–0.990). The AUC of an increased H-score of CYP11B1 (>204) for the diagnosis of CPA was 0.725 (95%CI 0.658–0.786). The AUC of a decreased H-score of CYP11B1 (

Context Familial hyperaldosteronism type I (FH-I) or glucocorticoid-remediable aldosteronism (GRA) is caused by unequal crossing over of the steroid 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes. Somatic KCNJ5 mutations have not been reported in patients with GRA; therefore, the appropriate treatment and prognosis of such concurrent cases remain unknown. Case Description Two siblings of a Taiwanese family with GRA were found to have adrenal adenomas and somatic KCNJ5 mutations. Complete clinical cure was achieved after unilateral adrenalectomy. Furthermore, the conversion site of the chimeric gene was identified by direct sequencing. Conclusions We report the coexistence of a somatic KCNJ5 mutation and GRA. Patients with GRA whose blood pressure management develops resistance to glucocorticoid treatment could therefore benefit from a lateralization test. The promising outcomes after unilateral adrenalectomy presented in this report offer new perspectives for further research into various PA subtypes.

Context Steroid hormones are important regulators of physiological processes in humans and are under genetic control. A link to coronary artery disease (CAD) is supposed. Objective Our main objective was to identify genetic loci influencing steroid hormone levels. As a secondary aim, we searched for causal effects of steroid hormones on CAD. Design We conducted genome-wide meta-association studies for eight steroid hormones: cortisol, dehydroepiandrosterone sulfate (DHEAS), estradiol, and testosterone in two independent cohorts (LIFE-Adult, LIFE-Heart, maximum n = 7667), and progesterone, 17-hydroxyprogesterone, androstenedione, and aldosterone in LIFE-Heart only (maximum n = 2070). All genome-wide significant loci were tested for sex interactions. Furthermore, we tested whether previously reported CAD single-nucleotide polymorphisms were associated with our steroid hormone panel and investigated causal links between hormone levels and CAD status using Mendelian randomization (MR) approaches. Results We discovered 15 novel associated loci for 17-hydroxyprogesterone, progesterone, DHEAS, cortisol, androstenedione, and estradiol. Five of these loci relate to genes directly involved in steroid metabolism, that is, CYP21A1, CYP11B1, CYP17A1, STS, and HSD17B12, almost completing the set of steroidogenic enzymes with genetic associations. Sexual dimorphisms were found for seven of the novel loci. Other loci correspond, for example, to the WNT4/β-catenin pathway. MR revealed that cortisol, androstenedione, 17-hydroxyprogesterone, and DHEA-S had causal effects on CAD. We also observed enrichment of cortisol and testosterone associations among known CAD hits. Conclusion Our study greatly improves insight into genetic regulation of steroid hormones and their dependency on sex. These results could serve as a basis for analyzing sexual dimorphism in other complex diseases.

Data on the contribution of gene polymorphism of proteins and enzymes of aldosterone metabolism to arterial hypertension are considered. On the basis of the analysis of scientific papers (eLIBRARY.RU, PubMed) and open databases (NCBI, GeneCard, QIAGEN, and related resources), the current status of ideas about the aldosterone metabolism genes (STAR, CYP11A1, HSD3B1, CYP21A2, CYP11B1, CYP11B2, UGT2B7), their expression, polymorphism, and the effect of the genetic variability on the encoded product was assessed. The role of the balance of aldosterone precursors in the regulation of blood pressure and the mutual influence of mineralocorticoids during aldosterone biogenesis on the final product of the reaction were analyzed. The main discussions on the studied issue were noted.

Hirsutism is a medical sign rather than a disease affects 5–8% of women of reproductive age. Hirsutism is associated with hyperandrogenemia in most patients excluding those with idiopathic hirsutism (IH). The most common cause of hirsutism is polycystic ovary syndrome (PCOS) followed by IH and idiopathic hyperandrogenemia (IHA); however, the clinical presentation of non-classical congenital adrenal hyperplasia (NCAH) in females is often indistinguishable from other hyperandrogenic disorders with common clinical signs such as hirsutism. The primary aim of the study is to examine the physical properties of the three genes and to make a detailed comparison of the mutations with the clinical data to contribute the etiology of hirsutism. 122 women admitted to the Endocrinology Clinic at Erciyes University Hospital with hirsutism were enrolled in the study between 2013-2014. All the participants were clinically evaluated. Protein-encoding exons, exon-intron boundaries of CYP21A2 (including proximal promoter), CYP11B1 and HSD3B2 genes were analyzed via state-of-the-art genetic studies. DNA sequencing analyses revealed two homozygous and three compound heterozygous 21-hydroxylase deficient (21OHD) NCAH patients. Additionally, three novel CYP21A2 mutations (A89V, M187I and G491S) and two novel CYP11B1 mutations (V188I and G87A) were determined. The frequencies of heterozygous mutations in CYP21A2 (including promoter), CYP11B1 and HSD3B2 genes were determined as 26.5% (15% coding region, 11.5% promoter), 11.5% and 0%, respectively. 21OHD-NCAH prevalence was determined to be ~4%. Unexpectedly, high heterozygous mutation rates were observed in CYP11B1 gene and CYP21A2 promoter region. CYP11B1 and HSD3B2 deficiencies were not prevalent in Turkish women with hirsutism despite the existence of higher heterozygous mutation rate in CYP11B1.

ACTH-independent Cushing’s syndrome (CS) is mainly caused by cortisol-secreting adrenocortical tumours. It is well known that secondary adrenal insufficiency occurs after surgical resection of these tumours. In this regard, impaired adrenocortical function is likely induced by atrophy of the residual adrenal tissue as a result of chronic suppression by the low ACTH levels of the hypercortisolism state. Therefore, we considered the prevention of adrenal atrophy as a method for preventing postoperative adrenal insufficiency. On the basis of these findings, we hypothesized that the use of a glucocorticoid receptor (GR) antagonist before surgery in ACTH-independent CS would rapidly activate the hypothalamic-pituitary-adrenal (HPA) axis and residual adrenal function. We thus examined adrenal function in a dexamethasone- (DEX-) induced CS rat model with or without mifepristone (MIF). In this study, MIF-treated rats had elevated plasma ACTH levels and increased adrenal weights. In addition, we confirmed that there were fewer atrophic changes, as measured by the pathological findings and mRNA expression levels of corticosterone synthase CYP11B1 in the adrenal glands, in MIF-treated rats. These results indicate that MIF treatment prevents the suppression of the HPA axis and the atrophy of the residual adrenal tissue. Therefore, our study suggests that preoperative GR antagonist administration may improve residual adrenal function and prevent postoperative adrenal insufficiency in ACTH-independent CS.

Human cytochrome P450 11B1 (CYP11B1) is responsible for the final step generating the steroid hormone cortisol, which controls stress and immune responses and glucose homeostasis. CYP11B1 is a promising drug target to manage Cushing's disease, a disorder arising from excessive cortisol production. However, the design of selective inhibitors has been hampered because structural information for CYP11B1 is unavailable and the enzyme has high amino acid sequence identity (93%) to a closely related enzyme, the aldosterone-producing CYP11B2. Here we report the X-ray crystal structure of human CYP11B1 (at 2.1 Å resolution) in complex with fadrozole, a racemic compound normally used to treat breast cancer by inhibiting estrogen-producing CYP19A1. Comparison of fadrozole-bound CYP11B1 with fadrozole-bound CYP11B2 revealed that despite conservation of the active-site residues, the overall structures and active sites had structural rearrangements consistent with distinct protein functions and inhibition. Whereas fadrozole binds to both CYP11B enzymes by coordinating the heme iron, CYP11B2 binds to the R enantiomer of fadrozole, and CYP11B1 binds to the S enantiomer, each with distinct orientations and interactions. These results provide insights into the cross-reactivity of drugs across multiple steroidogenic cytochrome P450 enzymes, provide a structural basis for understanding human steroidogenesis, and pave the way for the design of more selective inhibitors of each human CYP11B enzyme.

The adrenal gland is the most common toxicological target of drugs within the endocrine system, and inhibition of adrenal steroidogenesis can be fatal in humans. However, methods to evaluate the adrenal toxicity are limited. The aim of the present study was to verify the usefulness of simultaneous measurement of blood levels of multiple adrenal steroids, including precursors, as a method to evaluate drug effects on adrenal steroidogenesis in cynomolgus monkeys. With this aim, physiological and drug-induced changes in blood levels of adrenal steroids, including cortisol, aldosterone, androgen, and their precursors were examined. First, for physiological changes, intraday and interday changes in blood steroid levels were examined in male and female cynomolgus monkeys. The animals showed circadian changes in steroid levels that are similar to those in humans, while interday changes were relatively small in males. Next, using males, changes in blood steroid levels induced by ketoconazole and metyrapone were examined, which suppress adrenal steroidogenesis via inhibition of CYP enzymes. Consistent with rats and humans, both ketoconazole and metyrapone increased the deoxycorticosterone and deoxycortisol levels, probably via CYP11B1 inhibition, and the increase was observed earlier and with greater dynamic range than the changes in cortisol level. Changes in other steroid levels reflecting the drug mechanisms were also observed. In conclusion, this study showed that in cynomolgus monkeys, simultaneous measurement of blood levels of adrenal steroids, including precursors, can be a valuable method to sensitively evaluate drug effects on adrenal steroidogenesis and to investigate the underlying mechanisms.

Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency (21OHD) has a worldwide incidence of 1 in 15–20,000. Affected individuals have adrenal insufficiency and androgen excess; the androgen excess begins during fetal life, typically resulting in 46,XX disordered sexual development. In 21OHD, 17-hydroxyprogesterone (17OHP), the steroid proximal to 21-hydroxylase, accumulates. Most industrialized countries have newborn screening programs that measure 17OHP; such screening has permitted rapid detection of newborns with 21OHD, saving lives previously lost to mineralocorticoid deficiency and salt wasting. However, newborn screening is plagued by false positives. 17OHP is above most “cutoff values” in the first 24 h of life, is high in otherwise normal premature infants, and in many term infants with physiologic stress from unrelated diseases. In addition, newborn 17OHP may be elevated in other forms of CAH, including 11-hydroxylase deficiency, 3β-hydroxysteroid dehydrogenase deficiency, and P450 oxidoreductase deficiency. In 21OHD, some of the accumulated intra-adrenal 17OHP is converted to 21-deoxycortisol (21-deoxy) by 11β-hydroxylase (CYP11B1); 21-deoxy is not elevated in premature infants or in other forms of CAH, and hence is a more specific marker for 21OHD. However, 21-deoxy assays have not been generally available until recently, hence experience is limited. We urge clinical investigators, commercial reference laboratories, and newborn screening programs to investigate replacing 17OHP with 21-deoxy as the analyte of choice for studies of 21OHD.

Cushing's syndrome (CS) is a metabolic disorder caused by chronic hypercortisolism. CS is associated with cardiovascular, metabolic, skeletal and psychological dysfunctions and can be fatal if left untreated. The first-line treatment for all forms of CS is a surgery. However, medical therapy has to be chosen if surgical resection is not an option or is deemed ineffective. Currently available therapeutics are either not selective and have side effects or are only available as an injection (pasireotide). Areas covered: The authors discuss the recent drug developments for the medical treatment of CS through two validated molecular targets. Specifically, the authors look at selective inhibitors of CYP11B1 that reduce cortisol production by inhibiting steroid 11beta-hydroxylase and glucocorticoid receptor (GR) antagonists that interrupt cortisol-mediating transcriptional regulation of related genes. Expert opinion: Patients with CS have limited treatment options; indeed, there is an unmet need for new compounds that target CYP11B1 selectively versus several steroidogenic enzymes and/or GR-signaling pathways. The complexity of steroid biosynthesis and signaling requires the application of structure-based drug discovery techniques that use molecular targets and highly similar off-targets. Significant differences in steroidogenesis between humans and other species necessitates caution over the choice of in vivo model for the preclinical evaluation of future potential compounds.

Angiotensin II (Ang II: a truncated octapeptide of angiotensinogen, AGT) and 11-β-hydroxylase influence regulation of blood pressure. Dysregulation of Ang II and 11-β-hydroxylase can lead to hypertension and elevate aldosterone levels. Polymorphisms in AGT (encodes AGT) and CYP11B1 (encodes 11-β-hydroxylase) shift the paradigm from physiological to pathological. Currently, various high-throughput techniques are used to genotype these polymorphisms. These techniques require expensive infrastructure and reagents. However, in developing countries, where cost is the main limiting factor, it is not feasible to use expensive techniques. So, the aim of current study was to develop efficient low-cost method for genotyping of cardiovascular disease and hypertension associated polymorphisms of AGT (rs4762, rs5051) and CYP11B1 (rs6410). For this, tetra amplification-refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method was developed and optimized for aforementioned AGT and CYP11B1 gene polymorphisms. Efficiency of T-ARMS-PCR was tested by genotyping 776 human samples. These T-ARMS-PCR assays were also validated by Sanger DNA sequencing, where 100% concordance was found, allowing the efficient use of these T-ARMS-PCR assays for polymorphism genotyping in AGT and CYP11B1 in resource limited settings. T-ARMS-PCR is low-cost, efficient and reliable assay for genotyping of AGT and CYP11B1 gene polymorphisms.

BackgroundSomatic gene mutations that facilitate inappropriate intracellular calcium entrance have been identified in most aldosterone-producing adenomas (APAs). Studies suggest that angiotensin II and adrenocorticotropic hormone (ACTH) augment aldosterone production from APAs. Little is known, however, regarding possible variations in response to hormonal stimuli between APAs with different aldosterone-driver mutations.ObjectiveTo analyze the transcript expression of type 1 angiotensin II receptors (AGTR1), ACTH receptors (MC2R), and melanocortin 2 receptor accessory protein (MRAP) in APAs with known aldosterone-driver somatic mutations.MethodsRNA was isolated from APAs with mutations in: KCNJ5 (n = 14), ATP1A1 (n = 14), CACNA1D (n = 14), and ATP2B3 (n = 5), and from normal adjacent adrenal tissue (n = 45). Transcript expression of MC2R, MRAP, AGTR1, aldosterone synthase (CYP11B2), 17α-hydroxylase/17,20-lyase (CYP17A1), and 11β-hydroxylase (CYP11B1) were quantified using quantitative RT-PCR and normalized to β-actin.ResultsCompared to adjacent normal adrenal tissue, APAs had higher transcript levels of CYP11B2 (2,216.4 [1,112.0, 2,813.5]-fold, p < 0.001), MC2R (2.88 [2.00, 4.52]-fold, p < 0.001), and AGTR1 (1.80 [1.02, 2.80]-fold, p < 0.001]), and lower transcript levels of MRAP, CYP17A1, and CYP11B1 (0.28–0.36, p < 0.001 for all). MC2R and CYP11B2 transcripts were lower in APAs with KCNJ5 vs. other mutations (p < 0.01 for both). MC2R expression correlated positively with that of AGTR1 in APAs harboring KCNJ5 and CACNA1D mutations, and with MRAP expression in APAs harboring ATPase mutations.ConclusionsWhile MC2R and AGTR1 are expressed in all APAs, differences were observed based on the underlying aldosterone-driver somatic mutations. In tandem, our findings suggest that APAs with ATPase-mutations are more responsive to ACTH than KCNJ5-mutated APAs.

Keratinocytes control tissue homeostasis and skin inflammation via immunosuppressive glucocorticoids., Glucocorticoids (GC), synthesized by the 11β-hydroxylase (Cyp11b1), control excessive inflammation through immunosuppressive actions. The skin was proposed to regulate homeostasis by autonomous GC production in keratinocytes. However, their immunosuppressive capacity and clinical relevance remain unexplored. Here, we demonstrate the potential of skin-derived GC and their role in the regulation of physiological and prevalent inflammatory skin conditions. In line with 11β-hydroxylase deficiency in human inflammatory skin disorders, genetic in vivo Cyp11b1 ablation and long-term GC deficiency in keratinocytes primed the murine skin immune system resulting in spontaneous skin inflammation. Deficient skin GC in experimental models for inflammatory skin disorders led to exacerbated contact hypersensitivity and psoriasiform skin inflammation accompanied by decreased regulatory T cells and the involvement of unconventional T cells. Our findings provide insights on how skin homeostasis and pathology are critically regulated by keratinocyte-derived GC, emphasizing the immunoregulatory potential of endogenous GC in the regulation of epithelial immune microenvironment.

Congenital adrenal hyperplasia (CAH) is a group of disorders characterized by enzymatic defects in cortisol biosynthesis. Steroidogenic enzymes can mainly be divided into two groups: cytochrome P450 (CYP) and hydroxysteroid dehydrogenases/ketosteroid reductase (HSDs/KSR). The CYP enzyme group can metabolize more than one substrate and is involved in multiple steps of steroidogenesis. These enzymes have unidirectional function, catalyzing irreversibly the reactions they involved, and have no feedback from downstream precursors. The HSD enzymes catalyze reversible reactions, they modulate the steroid flux in the reductive or oxidative mode. Based on their activity, HSD enzymes are classified into dehydrogenases which oxidate hydroxysteroids to ketosteroids and reductases which reduce ketosteroids to hydroxysteroids. Due to mutations on these enzymes, one or more of the glucocorticoid, mineralocorticoid, and androgen biosynthesis pathways are affected. In all types of CAH, low cortisol production stimulate the secretion of corticotrophin releasing hormone (CRH) and adrenocorticotrophic hormone (ACTH) which leads to adrenal hyperplasia and eventually accumulation of steroid hormone precursors and usually hyperandrogenemia. The most common two forms are CYP21A2 (21-hydroxylase, 95% of cases) and CYP11B1 (11-beta hydroxylase, 5–8% of cases) deficiencies. Rare forms of CAH including 17-alpha hydroxylase and 3-beta hydroxysteroid dehydrogenase type 2 also affect gonadal steroid biosynthesis. Steroidogenic acute regulatory protein (StAR) deficiency and side chain cleavage enzyme deficiencies have similar clinical and laboratory characteristics except having lipoid accumulation in the former. P450 oxydoreductase deficiency is the most complicated form of CAH, and the clinical picture is a combination of 17-alpha hydroxylase and 21-hydroxylase deficiency.

Nonclassical congenital adrenal hyperplasia (NCAH) is a relatively common autosomal recessive disorder that arises from mutations in the genes encoding 21-hydroxylase (CYP21A2), 11-β-hydroxylase (CYP11B1), or 3-β-hydroxysteroid dehydrogenase (3βHSD2). Genetically confirmed nonclassical forms of 11-β-hydroxylase or 3-β-hydroxysteroid dehydrogenase deficiencies are extremely rare; the vast majority of subjects diagnosed with NCAH have mutations in both alleles of CYP21A2 that result in an overall 30–50% reduction in the activity of 21-hydroxylase enzyme. We will here review current challenges in the diagnosis of nonclassical 21-hydroxylase deficiency (21-OHd), including the reasons that justify screening for this disorder, the clinical situations in which such screening is required, recent advances in the methods used in different settings, and the consequences that might arise from a diagnosis of 21-OHd NCAH.

Glucocorticoid-remediable aldosteronism (GRA) is an autosomal-dominant inherited aldosteronism that is often accompanied by early-onset hypertension. GRA is caused by the unequal crossover of the 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes. As a result of chimeric gene duplication, aldosterone is ectopically synthesized in the adrenal zona fasciculata under the control of adrenocorticotropic hormone (ACTH). Here, we describe a Chinese pedigree with three affected subjects. Both the uncle and nephew were hospitalized in our hospital due to early-onset hypertension (onset

Background Cumulative exposure to high glucocorticoid levels is detrimental for the brain and may have particular implications in later life. A feature of late-life depression is increased cortisol secretion. Variants in the CYP11B1 gene, which codes for the enzyme responsible for cortisol synthesis, could influence risk of late-life depression, but this hypothesis has not been examined. We investigated the associations between variants in the CYP11B1 gene and late-life depression, taking into account history of depression and potential sex-specific effects. Methods We assessed depression in 1007 community-dwellers aged 65 years or older (60% women) at baseline and over a 14-year follow-up. A clinical level of depression was defined as a score of ≥ 16 on the Centre for Epidemiology Studies Depression scale or a diagnosis of current major depression based on the Mini-International Neuropsychiatric Interview and according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV). We examined incident and recurrent depression in participants without or with a history of major depression, respectively. We genotyped 5 single-nucleotide polymorphisms (SNPs) spanning CYP11B1. We used multivariable analyses to adjust for age, body mass index, cardiovascular ischemic pathologies, hypertension, cognitive impairment and anxiety. Results In women, rs6471580 and rs7016924 were associated with a 50% lower rate of incident (new-onset) late-life depression, and rs11783855 was associated with a 2.4-fold higher rate of late-life depression. These associations remained after correction for multiple testing, but we found no associations for recurrent depression in women or men. Limitations This study focused on the major gene involved in corticosteroid biosynthesis, but other genes may also be implicated in this pathway. Conclusion Variants of the CYP11B1 gene appear to be susceptibility factors for late-life depression in a sex-specific manner.

Background Given the rarity of 11β-hydroxylase deficiency (11βOHD), there is a paucity of data about the differences in clinical and biochemical characteristics of classic (C-11βOHD) and nonclassic 11βOHD (NC-11βOHD). Objective To characterize a multicenter pediatric cohort with 11βOHD. Method The clinical and biochemical characteristics were retrospectively retrieved. CYP11B1 gene sequencing was performed. Seventeen plasma steroids were quantified by liquid chromatography-mass spectrometry and compared to that of controls. Results 102 patients (C-11βOHD, n = 92; NC-11βOHD, n = 10) from 76 families (46,XX; n = 53) had biallelic CYP11B1 mutations (novel 9 out of 30). Five 46,XX patients (10%) were raised as males. Nineteen patients (19%) had initially been misdiagnosed with 21-hydroxylase deficiency. Female adult height was 152 cm [−1.85 SD score (SDS)] and male 160.4 cm (−2.56 SDS).None of the NC-11βOHD girls had ambiguous genitalia (C-11βOHD 100%), and none of the NC-11βOHD patients were hypertensive (C-11βOHD 50%). Compared to NC-11βOHD, C-11βOHD patients were diagnosed earlier (1.33 vs 6.9 years; P 2.2, Conclusion NC-11βOHD can escape from clinical attention due to relatively mild clinical presentation. However, steroid profiles enable the diagnosis, differential diagnosis, and subtyping of 11βOHD.

Objective To report clinical, hormonal and structural effects of CYP11B1 pathogenic variations in Indian patients with 11β-hydroxylase deficiency (11βOHD) and find hormonal criteria that accurately distinguish 11βOHD from 21α-hydroxylase deficiency (21OHD). Design Retrospective record review of genetically diagnosed patients with 11βOHD. Patients and measurements Clinical features, hormonal parameters at diagnosis (by immunoassay) and recent follow-up of 13 genetically proven 11βOHD patients managed at our centre were retrospectively reviewed. ACTH-stimulated serum adrenal steroids (measured by LC-MS/MS) of 11βOHD were compared with those of simple virilizing and non-classic 21OHD. Structural analysis of the observed pathogenic variations was performed by computational modelling. Results Nine (four females) and four (all females) patients had classic and non-classic disease, respectively. All 11βOHD patients had elevated ACTH-stimulated serum 11-deoxycortisol (26.5-342.7 nmol/L) whereas none had elevated serum 17-hydroxyprogesterone (4.2-21.2 nmol/L); both hormonal parameters distinguished 11βOHD from 21OHD with 100% accuracy. ACTH-stimulated serum cortisol, but not 11-deoxycortisol, clearly distinguished classic ( 160 nmol/L) disease. Thirteen (eight novel, two recurrent) pathogenic variants were observed. Only missense mutations were observed among patients with non-classic disease. Computational modelling predicted the possible affection of enzyme structure and function for all the observed missense mutations. Conclusions This first Indian study describes 13 11βOHD patients, including four with the rarer non-classic variant. A total of eight novel pathogenic variants were identified in our study, highlighting regional genetic heterogeneity. Measurement of ACTH-stimulated adrenal steroids by LC-MS/MS will help avoid the misdiagnosis of 11βOHD as 21OHD and has potential to distinguish classic from non-classic 11βOHD.

Although cytochrome P450 (CYP) systems including the adrenal ones are being investigated since many years, there are still reactions and regulation patterns that have been underestimated ever since. This review discusses neglected ones to bring them into the focus of investigators working in the field. Novel substrates and reactions described for adrenal CYPs recently point to the fact that different from what has been believed for many years, adrenal CYPs are less selective than previously thought. The conversion of steroid sulfates, intermediates of steroid biosynthesis as well as of exogenous compounds are being discussed here in more detail and consequences for further studies are drawn. Furthermore, it was shown that protein-protein interactions may have an important effect not only on the activity of adrenal CYPs, but also on the product pattern of the reactions. It was found that, as expected, the stoichiometry of CYP:redox partner plays an important role for tuning the activity. In addition, competition between different CYPs for the redox partner and for electrons and possible alterations by mutants in the efficiency of electron transfer play an important role for the activity and product pattern. Moreover, the influence of phosphorylation and small charged molecules like natural polyamines on the activity of adrenal systems has been demonstrated in-vitro indicating a possible regulation of adrenal CYP reactions by affecting redox partner recognition and binding affinity. Finally, an effect of the genetic background on the consequences of mutations in adrenal CYPs found in patients was suggested from corresponding in-vitro studies indicating that a different genetic background might be able to significantly affect the activity of a CYP mutant.

Steroidogenesis is strictly regulated at multiple levels, as produced steroid hormones are crucial to maintain physiological functions. Cytochrome P450 enzymes are key players in adrenal steroid hormone biosynthesis and function within short redox-chains in mitochondria and endoplasmic reticulum. However, mechanisms regulating supply of reducing equivalents in the mitochondrial CYP-dependent system are not fully understood. In the present work, we aimed to estimate how the specific steroids, substrates, intermediates and products of multistep reactions modulate protein-protein interactions between adrenodoxin (Adx) and mitochondrial CYP11s. Using the SPR technology we determined that steroid substrates affect affinity and stability of CYP11s – Adx complexes in an isoform-specific mode. In particular, cholesterol induces a 4-fold increase in the rate of CYP11A1 – Adx complex formation without significant effect on dissociation (koffdecreased ~1.5-fold), overall increasing complex affinity. At the same time steroid substrates decrease the affinity of both CYP11B1 – Adx and CYP11B2 – Adx complexes, predominantly reducing their stability (4-7 fold). This finding reveals differentiation of protein-protein interactions within the mitochondrial pool of CYPs, which have the same electron donor. The regulation of electron supply by the substrates might affect the overall steroid hormones production. Our experimental data provide further insight into protein-protein interactions within CYP-dependent redox chains involved in steroidogenesis.

Congenital adrenal hyperplasia refers to a group of rare genetic disorders affecting the adrenal glands. 21-hydroxylase deficiency is the most prevalent and the most studied cause while the remaining enzymatic defects are less common, accounting for less than 10% of cases. We herein described the clinical, biological and molecular characteristics and outcome of patients of the same family diagnosed with 11-Beta-hydroxylase deficiency. The disorder was revealed by peripheral precocious puberty between the age of 2-3 years in males and by the virilization of the external genitalia in females. Genetics finding a homozygous p.Gly379Val mutation in the CYP11B1 gene. All patients received hydrocortisone supplementation therapy and mineralocorticoid-receptor antagonist. The females underwent a surgical correction of the ambiguous genitalia at the neonatal age. Long term follow-up revealed metabolic syndrome, obesity and hypertension in the first two patients, an impaired final height in the two females and hypokalemia in three patients.

Objective: 11β-hydroxylase deficiency (11βOHD) is a rare autosomal recessive disorder caused by mutations in the CYP11B1 gene. It is characterized by virilization, hypertension, and significant final height impairment. In this study, we aim to investigate the clinical and molecular characteristics of four unrelated Chinese patients with 11βOHD disorder.Methods: The clinical information of four 11βOHD patients were carefully reviewed. Genetic analysis was performed using next-generation sequencing (NGS) based panel analysis. NGS coverage depth was analyzed to detect exonic copy-number variants (CNVs) on patient 1. Quantitative PCR (qPCR) was subsequently performed to confirm the CNVs detected from the NGS coverage depth analysis.Results: The mean age of the patients at diagnosis was 4.7 years (range, 2.0–9.3 years). Two genetically female patients (patients 1 and 2) with 11βOHD presented severe virilization of external genitalia and were raised as males. Two genetically male patients (patients 3 and 4) presented precocious puberty. Additionally, patients 1, 3, and 4 presented with hypertension. In patient 4, unilateral adrenal mass was detected and removed at the age of 9 years. Interestingly, the height of patient 4 (174.4 cm, +6.7 SD) wasn't impaired and reached his mid-parental height (173 cm). Three novel variants in the CYP11B1 gene (c.1150_1153del, c.217C>T, and c.400G>C) were identified by NGS. Various bioinformatics tools revealed potential pathogenic effects for the novel variants, and evolutionary-conservation revealed that the novel missense variant affected an amino acid that is highly conserved among species. Furthermore, NGS coverage depth analysis and qPCR identified a novel heterozygous deletion of exons 1–6 in patient 1.Conclusion: Our study expands the spectrum of mutations of the CYP11B1 gene in Chinese population. In addition, We reported the first case of a patient with classical 11βOHD disorder, whose final height wasn't compromised.

Context Isolated congenital hypoaldosteronism presents in early infancy with symptoms including vomiting, severe dehydration, salt wasting, and failure to thrive. The main causes of this rare autosomal recessive disorder is pathogenic variants of the CYP11B2 gene leading to aldosterone synthase deficiency. Objective To investigate the presence of CYP11B2 pathogenic variants in a cohort of patients with a clinical, biochemical, and hormonal profile suggestive of aldosterone synthase deficiency. Design Clinical and molecular study. Setting Tertiary academic Children’s Hospital, Center for Rare Pediatric Endocrine Diseases. Patients and Methods Sixty-two patients (56 unrelated patients and 6 siblings), with hypoaldosteronism and their parents, underwent CYP11B2 gene sequencing after its selective amplification against the highly homologous CYP11B1 gene. In silico analysis of the identified novel variants was carried out to evaluate protein stability and potential pathogenicity. Results CYP11B2 gene sequencing revealed that 62 patients carried a total of 12 different pathogenic CYP11B2 gene variants, 6 of which are novel. Importantly, 96% of the 56 patients carried the previously reported p.T185I variant either in homozygosity or in compound heterozygosity with another variant. The 6 novel variants detected were: p.M1I, p.V129M, p.R141Q, p.A165T, p.R448C, and the donor splice site variant of intron 8, c.1398 + 1G > A. Conclusion Molecular diagnosis was achieved in 62 patients with aldosterone synthase deficiency, the largest cohort thus far reported. Six novel genetic variants were identified as possibly pathogenic, extending the spectrum of reported molecular defects of the CYP11B2 gene.

Testicular Adrenal Rest Tumors (TART) are a common complication of unknown origin in patients with Congenital Adrenal Hyperplasia. These benign tumors may derive from cells of adrenal origin or from pluripotent progenitor cells from the fetal adrenogonadal primordium. By comparing the transcriptome of TART with fetal- and adult-testis and adrenal tissues, this study aims to unravel the origin of TART. Targeted transcriptome sequencing was followed by unsupervised clustering-, differential expression-, functional enrichment- and pathway analyses. Immunohistochemistry demonstrated co-expression of adrenal-specific CYP11B1 and testis-specific HSD17B3 in TART, indicating the existence of a distinct TART cell exhibiting both adrenal- and testicular characteristics. Principal component analysis indicated that the TART transcriptome was distinct from either adrenal or testis fetal tissue, making a progenitor-like phenotype of TART unlikely. Rather, TART appears to originate from -or differentiate into-a mature cell type, with both adrenal- and testicular characteristics. The present study, the first to describe the TART transcriptome, expands knowledge about the identity and functional characteristics of TART and identifies clinically targetable pathways associated with fibrosis.

Introduction: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder, caused by a deficiency in one of the enzymes involved in adrenal steroid synthesis. Homozygotes usually have a severe classical CAH phenotype. Heterozygotes, carrying only one abnormal copy of the gene, are thought to be generally asymptomatic, although could be associated with hyperandrogenism, decreased fertility, adrenal incidentalomas. 21-hydroxylase deficiency (21OHD) accounts for 90% of all CAH cases, while 11 β-hydroxylase deficiency (11OHD) accounts for 4–8% of CAH cases. The nature and mechanism of a combined enzymatic defect are unknown. The coincidental presence of gene mutation for both 21OHD and 11OHD CAH in a single individual is very unlikely to occur. Clinical case: A 22-year old female with no significant past medical history presented to endocrinologist for evaluation of facial hirsutism. Patient had menarche at age 11, and menstrual cycle was regular since. No concerns for virilization of external genitalia. She was not sexually active, no pregnancies. No Family history of infertility or genetic conditions. Patient’s father was Jewish, and mother was Slavic. Physical examination revealed female phenotype, normal Blood pressure and BMI, acne on the back and upper arm, Ferriman-Gallwey hirsutism score 5. Labs: AM cortisol, CMP, CBC and TSH were normal. Total testosterone 68 ng/dL (2–45), free testosterone 7 pg/mL (0.1 - 6.4), FSH 5.7 mIU/mL (2.5–10.2), LH 10.6 mIU/mL (1.9–12.5), Progesterone 2.1 ng/mL ( Elevated 11-Deoxycortisol level raised a suspicion for 11-OHD CAH, or adrenal vs ovarian hormone-producing mass. Abdominal CT and pelvic US were negative for adrenal or ovarian masses. 3-day dexamethasone suppression test completely normalized all biochemical abnormalities the patient had. Genetic testing showed: CYP21A2 c.844G>T (non-classic 21OHD CAH mutation), CYP21A2 c.923dupT (classic 21OHD CAH mutation), CYP11B1 c.953C>G mutation. Thus diagnosis of non-classic 21OHD CAH, and carrier status of 11OHD CAH was made. She was started on oral Dexamethasone 0.25 mg every other day. 11-Deoxycortisol elevation could not be explained by 21OHD alone. Her carrier state of the CYP11B1 mutation also cannot cause elevated 11-Deoxycortisol level. We hypothesize that 11-Deoxycortisol was elevated either from extra adrenal conversion of 17-hydroprogesterone to 11-Deoxycortisol, or from 11 β-hydroxylase inhibition by excess intra-adrenal androgens. Conclusion: Our case reports a rare finding of both CYP21A2 and CYP11B1 mutations in the same individual, which has implications for relatives, family planning and partner genetic screening.

OBJECTIVE To investigate the association of genetic variants of renin angiotensin aldosterone system, endothelial nitric oxide synthase and 11-beta-hydroxylase genes, and the drug efficacy of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker. METHODS This two time-point study was conducted from April to November 2016 at Allied Hospital, Faisalabad and National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, and comprised of hypertensive patients taking angiotensin-converting enzyme inhibitor and angiotensin receptor blocker who were followed up for 12 weeks. Baseline and follow-up clinical and biochemical parameters were measured for all patients. Total 11 polymorphisms were genotyped by polymerase chain reaction, polymerase chain reaction-restriction fragment length polymorphism and amplification-refractory mutation system-polymerase chain reaction assays. Data was divided into baseline and follow-up groups, while the latter group was further divided into responding and non-responding subgroups on the basis of patient response to angiotensin-converting enzyme inhibitor and angiotensin receptor blocker drugs. Data was analysed using SPSS 20. RESULTS Of the 45 patients, 25(55.5%) were females and 20(44.5%) were males. There was a significant reduction in the systolic blood pressure (p=0.004) and low-density lipoprotein cholesterol (p

Primary aldosteronism (PA), also known as Conn’s syndrome, is a common curable cause of hypertension. Family studies of essential hypertensive patients suggest that heritable genetic factors play a role in blood pressure regulation1. Interestingly, single nucleotide polymorphisms (SNP) in genes encoding enzymes involved with adrenal steroidogenesis, CYP11B2, CYP11B1 and CYP17A1, associate with increased risk of hypertension2. Therefore, we analysed whether selected SNPs in these genes are associated with PA. We performed an association study using genotype imputation for selected SNPs of the steroidogenic enzyme genes CYP11B2 (rs4546, rs1799998, rs13268025), CYP11B1 (rs6410, rs149845727), and CYP17A1 (rs1004467, rs138009835, rs2150927) from a pilot genome wide association study of Malaysian PA patients and healthy controls which was merged with the Singapore Genome Variation Project (SGVP) population dataset3. Genotype imputation for minor and major alleles was validated using PCR sequencing (n>10 for each genotype). Further, one SNP from each steroidogenic enzyme (CYP11B2:rs1799998, CYP11B1:rs6410 and CYP17A1:rs1004467) was validated using commercial TaqMan genotyping assays on the ABI 7000 Sequence Detection System which was performed on 149 PA patients and 78 non-hypertensive healthy individuals. Case-control genetic association analysis was performed at http://www.oege.org/software/orcalc.html and the association between genotypes and phenotypes was done using the independent-samples Kruskal-Wallis test on SPSS (version 25). The Minor Allele Frequencies (MAFs) for rs1004467, rs6410 and rs1799998 were similar to East Asian populations but differed significantly different from European, African, American and South Asian populations (rs1004467 MAF: C=0.258/298, rs6410 MAF: A=0.265/298, rs1799998 MAF: C=0.225/298). In Chinese patients matched by gender, heterozygotes for rs6410 had significantly increased risk of PA compared to common homozygotes (OR: 3.15, 95% CI: 1.01–9.8, p=0.04). Across patients of different ethnicity, the distribution of aldosterone levels was significantly different (p=0.039). In summary, only SNP rs6410 in Chinese patients matched by gender showed association with PA in our South East Asian cohort. More functional experiments need to be done to find out whether this is causal for PA or whether the SNP is in linkage disequilibrium with the actual functional causative SNPs. Once the functional SNP is known, identification of these germline variants in asymptomatic family members would allow early screening of PA to be offered and potentially provide novel drug targets to treat the disease. References: 1Timberlake et al., Curr Opin Nephrol Hypertens. 2001 Jan;10(1):71-9. 2MacKenzie et al., Int J Mol Sci. 2017 Mar 7;18(3). pii: E579. 3Teo et al., Genome Res. 2009 Nov;19(11):2154-62.

Osilodrostat (Isturisa®) is an orally available small molecule 11β-hydroxylase inhibitor that is being developed by Novartis for the treatment of Cushing's disease. Based on results from a pivotal phase III trial, osilodrostat was approved in the EU for use in the treatment of endogenous Cushing's syndrome in adults and is under regulatory review in the USA for the treatment of Cushing's disease. This article summarises the milestones in the development of osilodrostat leading to this first approval.

Background The prevalence of primary aldosteronism concurrent with subclinical Cushing’s syndrome was higher than previously thought. Through analyzing a rare clinical case, we summarized the diagnosis and management of primary aldosteronism with subclinical Cushing’s syndrome. Case presentation A 54-year-old Chinese man of Han nationality was diagnosed as having primary aldosteronism with subclinical Cushing’s syndrome. An abdominal computed tomography scan revealed a mass in his left adrenal gland and a mass in his right adrenal gland. After finishing sequential adrenal venous sampling without adrenocorticotropic hormone, the result reminded us that the left and right nodules were responsible for hypercortisolism and aldosterone hypersecretion, respectively. Right and left adrenalectomy were performed successively. The pathological diagnosis was adrenocortical adenoma for both. Histological findings revealed that the right one had positive immunostaining for CYP11B2 and the left one had positive immunostaining for CYP11B1. The immunohistochemistry result helped us to confirm the diagnosis. Somatic KCNJ5 mutation (Leu168Arg) was found in the right tumor; there was no KCNJ5 mutation in the left adrenal tumor. Conclusions We suggest that patients with primary aldosteronism should have a low-dose overnight dexamethasone suppression test to screen for hypercortisolism. It can help avoid misdiagnoses and contribute to proper understanding of the adrenal vein sampling result. Making sure of the nidus of aldosterone and cortisol secretion is crucial for the therapy of patients with primary aldosteronism and subclinical Cushing’s syndrome.

Aldosterone-producing adenomas (APAs) are one of the main causes of primary aldosteronism and the most prevalent surgically correctable form of hypertension. Aldosterone-producing cell clusters (APCCs) comprise tight nests of zona glomerulosa cells, strongly positive for CYP11B2 (aldosterone synthase) in immunohistochemistry. APCCs have been suggested as possible precursors of APAs because they frequently carry driver mutations for constitutive aldosterone production, and a few adrenal lesions with histopathologic features of both APCCs and APAs have been identified. Our objective was to investigate the metabolic phenotypes of APCCs (n=27) compared with APAs (n=6) using in situ matrix-assisted laser desorption/ionization mass spectrometry imaging of formalin-fixed paraffin-embedded adrenals from patients with unilateral primary aldosteronism. Specific distribution patterns of metabolites were associated with APCCs and classified 2 separate APCC subgroups (subgroups 1 and 2) indistinguishable by CYP11B2 immunohistochemistry. Metabolic profiles of APCCs in subgroup 1 were tightly clustered and distinct from subgroup 2 and APAs. Multiple APCCs from the same adrenal displayed metabolic profiles of the same subgroup. Metabolites of APCC subgroup 2 were highly similar to the APA group and indicated enhanced metabolic pathways favoring cell proliferation compared with APCC subgroup 1. In conclusion, we demonstrate specific subgroups of APCCs with strikingly divergent distribution patterns of metabolites. One subgroup displays a metabolic phenotype convergent with APAs and may represent the progression of APCCs to APAs.

Introduction Tumor necrosis factor-α (TNF-α) plays an extremely important role in the regulation of hypothalamicpituitary-adrenal axis. It is believed that chronic inflammation is the main cause of cancerogenesis and TNF-α plays a significant role in both of these processes. Unfortunately, the function of TNF-α in human adrenal steroidogenesis has not been explained enough. Aim To evaluate the changes in transcriptional activity of STAR, CYP11A1, CYP11B1, and CYP11B2 in H295R cell line exposed to TNF-α. Material and methods NCI-H295R, human adrenocortical cell line was exposed to human recombinant TNF-α at the concentrations ranging from 0.001 to 10 nM for 3, 12, 24, and 48 h. Cells not exposed to TNF-α were the control of this experiment. RTqPCR assay was used to determine the changes in the expression of genes encoding STAR, CYP11A1, CYP11B1, and CYP11B2. Results The highest differences between stimulated and non-stimulated cells were observed in the expression of STAR (FC = +2.2; 0.01 nM of TNF-α; 48 h); CYP11A1 (FC = +3.5; 0.1 nM of TNF-α; 24 h); CYP11B1 (FC = +7.0; 10 nM of TNF-α; 48 h); CYP11B2 (FC = +2.5; 10 nM of TNF-α; 48 h). Statistically significant differences (p < 0.05) in the expression were found only for CYP11A1. The interaction effect between genes was also noticed (p < 0.05). Conclusions The research showed the impact of TNF-α on the expression of the key genes encoding enzymes involved in adrenal steroidogenesis. Different expression patterns of was observed, depending on time and TNF-α concentration increased synthesis of this pro-inflammatory cytokine may intensify adrenal steroidogenesis.

International audience; Aldosterone-producing adenoma (APA) cause primary aldosteronism—the most frequent form of secondary hypertension. Somatic mutations in genes coding for ion channels and ATPases are found in APA and in aldosterone-producing cell clusters. We investigated the genetic, cellular, and molecular heterogeneity of different aldosterone-producing structures in adrenals with APA, to get insight into the mechanisms driving their development and to investigate their clinical and biochemical correlates. Genetic analysis of APA, aldosterone-producing cell clusters, and secondary nodules was performed in adrenal tissues from 49 patients by next-generation sequencing following CYP11B2 immunohistochemistry. Results were correlated with clinical and biochemical characteristics of patients, steroid profiles, and histological features of the tumor and adjacent adrenal cortex. Somatic mutations were identified in 93.75% of APAs. Adenoma carrying KCNJ5 mutations had more clear cells and cells expressing CYP11B1, and fewer cells expressing CYP11B2 or activated β-catenin, compared with other mutational groups. 18-hydroxycortisol and 18-oxocortisol were higher in patients carrying KCNJ5 mutations and correlated with histological features of adenoma; however, mutational status could not be predicted using steroid profiling. Heterogeneous CYP11B2 expression in KCNJ5 -mutated adenoma was not associated with genetic heterogeneity. Different mutations were identified in secondary nodules expressing aldosterone synthase and in independent aldosterone-producing cell clusters from adrenals with adenoma; known KCNJ5 mutations were identified in 5 aldosterone-producing cell clusters. Genetic heterogeneity in different aldosterone-producing structures in the same adrenal suggests complex mechanisms underlying APA development.

Autonomous steroid secretion is a common feature of adrenocortical carcinomas (ACC), although not always clinically evident owing to inefficient steroidogenesis with increased release of steroid precursors. Our study aim was to analyze the expression profile of four key proteins involved in the steroidogenesis cascade, in different adrenocortical tumors. Expression of proteins involved in steroidogenesis, namely steroidogenic acute regulatory protein (StAR), 11&beta, hydroxylase (CYP11B1), aldosterone synthase (CYP11B2) and 17&alpha, hydroxylase (CYP17A1), were analyzed by immunohistochemistry in ACC (n = 14), adenomas presenting with Cushing&rsquo, s syndrome (ACAc) (n = 11) and clinically non-functioning adenomas (ACAn) (n = 15). A percentage of the stained area for each protein was analyzed using ImageJ software for computerized morphometric quantification. CYP11B1, StAR and CYP17A1 expression were significantly lower in ACC when compared to ACAc. In addition, ACC presented co-staining cells for CYP11B1 and CYP11B2. CYP11B1 was the steroidogenic enzyme with the most discriminative power to distinguish ACC from ACAc, with a sensitivity of 100%, specificity of 92%, and an expression higher than 4.44%, indicating the presence of a cortisol secreting adenoma. ACC depicts an incomplete pattern of steroidogenic protein expression, with decreased CYP11B1 and CYP17A1, which could explain the predominant secretion of steroid precursors.