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Background Testicular adrenal rest tumors (TART) are a common complication of unknown cellular origin in patients with congenital adrenal hyperplasia (CAH). These benign tumors have both adrenal and testicular characteristics and are hypothesized to either derive from cells of adrenal origin from the fetal adrenogonadal primordium or by atypical differentiation of adult Leydig-progenitor cells. Objective This study aims to unravel the identity and etiology of TART. Methods Co-expression of adrenal-specific CYP11B1 and Leydig cell-specific HSD17B3 in TART was studied using immunohistochemistry. We studied the possibility of TART being derived from atypical differentiation of adult Leydig-progenitor cells by the quantification of adrenal-specific enzyme expression upon adrenocorticotrophic hormone (ACTH)-like stimulation of ex vivo cultured platelet-derived growth factor receptor alpha-positive cells. By comparing the transcriptome of TART (n = 16) with the transcriptome of fetal adrenal (n = 13), fetal testis (n = 5), adult adrenal (n = 11), and adult testis (n = 10) tissues, we explored the identity of TART. Results We demonstrate co-expression of adrenal-specific CYP11B1 and testis-specific HSD17B3 in TART cells, indicating the existence of a distinct TART cell exhibiting both adrenal and testicular characteristics. Ex vivo cultured adult Leydig-progenitor cells did not express the ACTH-receptor MC2R but did express CYP11B1 upon stimulation. Unsupervised clustering of transcriptome data showed that TART was most similar to adult adrenal tissue, followed by adult testis tissue, and least similar to either fetal tissue. Conclusion Our data suggest that TART is induced — most likely via activation of a cAMP/protein kinase A-dependent receptor — from a progenitor cell into a unique mature adrenal-like cell type, sometimes exhibiting both adrenal and testicular features.

Introduction Osilodrostat is a new 11β-hydroxylase inhibitor with a mode of action analogous to Metyrapone. The objective of this study was to compare steroidogenic profiles in patients treated with either Osilodrostat or Metyrapone for adrenocorticotrophic hormone (ACTH)-dependent Cushing's syndrome (CS). Methods Patients followed up at Cochin hospital Endocrinology department between March 2019 and December 2021 for an ACTH-dependent CS, controlled by either Osilodrostat or Metyrapone, were included. A serum profile of five steroids (cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, androstenedione and testosterone) was determined using UPLC- tandem mass spectrometry (UPLC-MS/MS). Results Nineteen patients treated with Osilodrostat, eight patients treated with Metyrapone and six patients treated with consecutive Metyrapone then Osilodrostat were included. Hypocortisolism (basal cortisol Conclusion In patients with ACTH-dependent CS, the use of CYP11B1 inhibitors in routine care suggests that Osilodrostat has a less specific effect on the inhibition of steroidogenic enzymes than Metyrapone. This might explain a smaller increase in 11-deoxycortisol and androgen levels in patients treated with Osilodrostat.

Background Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder that has been included in newborn screening programs. Current approaches to gene testing for CAH are facing challenges because of the complexity of the CYP21A2 locus and genetic heterogeneity of the disease. Methods A comprehensive analysis of CAH (CACAH) combining long-range locus-specific PCR and long-read sequencing (LRS) was developed to perform full sequence analysis of 5 common CAH candidate genes, including CYP21A2, CYP11B1, CYP17A1, HSD3B2, and StAR. In a blind retrospective study, the clinical utility of CACAH was evaluated in 37 samples by comparing to standard CAH testing using multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing. Results Of the 37 clinical samples, a total of 69 pathogenic variants were identified, comprising 65 CYP21A2 variants, 2 HSD3B2 variants, and 2 CYP17A1 variants. For CYP21A2, the most frequent variant was c.518T > A (29.2%), followed by c.293−13C/A > G (21.5%). Compared with the current CAH testing using MLPA plus Sanger sequencing, the CACAH assay showed 100% specificity and 100% sensitivity, and precisely determined the junction sites of deletions/insertions and cis–trans configuration of multiple variants without analyzing family samples. Moreover, CACAH identified a case carrying 2 copies of CYP21A1 with the c.1451_1452delinsC variant on the same chromosome, which was not confirmed by MLPA plus Sanger sequencing. Conclusion LRS-based CACAH can determine all genotypes of CAH accurately and reliably in one assay, presenting a comprehensive approach for CAH genetic diagnosis and carrier screening.

Primary aldosteronism is considered the commonest cause of secondary hypertension. In affected individuals, aldosterone is produced in an at least partially autonomous fashion in adrenal lesions (adenomas, [micro]nodules or diffuse hyperplasia). Over the past decade, next-generation sequencing studies have led to the insight that primary aldosteronism is largely a genetic disorder. Sporadic cases are due to somatic mutations, mostly in ion channels and pumps, and rare cases of familial hyperaldosteronism are caused by germline mutations in an overlapping set of genes. More than 90% of aldosterone-producing adenomas carry somatic mutations in K + channel Kir3.4 ( KCNJ5 ), Ca 2+ channel Ca V 1.3 ( CACNA1D ), alpha-1 subunit of the Na + /K + ATPase ( ATP1A1 ), plasma membrane Ca 2+ transporting ATPase 3 ( ATP2B3 ), Ca 2+ channel Ca V 3.2 ( CACNA1H ), Cl − channel ClC-2 ( CLCN2 ), β-catenin ( CTNNB1 ), and/or G-protein subunits alpha q/11 ( GNAQ/11 ). Mutations in some of these genes have also been identified in aldosterone-producing (micro)nodules, suggesting a disease continuum from a single cell, acquiring a somatic mutation, via a nodule to adenoma formation, and from a healthy state to subclinical to overt primary aldosteronism. Individual glands can have multiple such lesions, and they can occur on both glands in bilateral disease. Familial hyperaldosteronism, typically with early onset, is caused by germline mutations in steroid 11-beta hydroxylase/ aldosterone synthase ( CYP11B1/2 ), CLCN2 , KCNJ5 , CACNA1H , and CACNA1D .

Prevalence of hypertension and hypertension-mediated organ damage (HMOD) had not been well studied in patients with 11β-hydroxylase deficiency (11β-OHD).The study was to assess the prevalence and risk factors of hypertension and HMOD in patients with 11β-OHD.Retrospective cohort analysis in a single medical centre.Twenty-eight patients with 11β-OHD were recruited between January 2003 and June 2021, and their diagnosis had been confirmed by Sanger sequencing.Blood pressure and clinical indicators for the assessment of HMOD occurrence were collected from the medical records. Medication adherence of antihypertensive drugs and glucocorticoids were determined by the patients' biochemistry. Logistic regression was used to identify factors associated with HMOD.Prevalence of hypertension and HMOD in the cohort was 100% and 50%, respectively. The kidneys (71.43%) are the organ most commonly damaged by high blood pressure, followed by the heart (64.29%), eyes (57.14%) and brain (21.43%). Risk factors of HMOD were hypokalemia (odds ratio [OR]: 9.16; 95% confidence interval [CI]: 1.634-51.43; p = .012), blood pressure ≥ 180/110 mmHg (OR: 22.0, 95% CI: 3.08-157.34; p = .002) and irregular glucocorticoid use (OR: 3.18, 95% CI: 1.13-8.98; p = .021). Blood pressure ≥ 180/110 mmHg was an independent predictor for HMOD.Hypertension and HMOD are prevalent in patients with 11β-OHD in our study. These findings illustrate the importance of early HMOD evaluation and optimal glucocorticoid medication in 11β-OHD patients.

Autonomous cortisol secretion (ACS) could be found in some patients with unilateral primary aldosteronism (uPA). However, the histopathological patterns of uPA with concurrent ACS have not been well elucidated. The adrenal gland with the adenoma from 61 uPA patients who underwent unilateral adrenalectomy were assessed by immunohistochemistry. Bioinformatics analysis, including the Cancer Genome Atlas (TCGA) and Kyoto Encyclopedia of Genes and Genomes, was applied. The prevalence of multiple aldosterone-producing nodules or micronodules (mAPN/mAPM) was 65.6% (40/61) among our uPA patients. Concurrent ACS was identified in 32% of this uPA cohort; they were associated with the interaction of larger tumor size (>1.98 cm) and mAPN/mAPM (odds ratio = 3.08, P = 0.004). Transcriptome analysis uncovered a dominant enrichment of HSD3B7 overexpression (P = 0.004) in the adenomas of the histopathologically classical adrenal uPA lesions with concomitant mAPN/mAPM, compared with those uPA adenomas without concurrent surrounding mAPN/mAPM. We identified a novel linkage of enhanced steroidogenic genes of HSD3B7 expression concurrent with the downstream higher CYP11B1 expression; further relationship was confirmed by immunohistochemical staining and validated by TCGA bioinformatics. The presence of mAPN/mAPM in uPA patients had lower rate for biochemical success after adrenalectomy (P = 0.047). In summary, two-thirds of uPA patients had concomitant mAPN/mAPM; 1/3 of uPA patients had concurrent ACS. Steroidogenic HSD3B7/CYP11B1 signaling was associated with uPA adenomas with surrounding mAPN/mAPM. Interaction of larger adenoma size with the presence of mAPN/mAPM was linked to co-existing ACS. Such uPA patients with concomitant mAPN/mAPM had lower rate of biochemical success.

Background Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by pathogenic variants in seven genes involved in the cortisol and aldosterone biosynthetic pathway. The second most common cause, 11β-hydroxylase deficiency (11βOHD), is attributed to pathogenic variants in the CYP11B1 gene encoding for the enzyme 11β-hydroxylase (11βOH). Case presentation A 13-year-old girl was referred to the pediatric endocrinologist due to a syncopal episode. She is the third child of non-consanguineous parents. She presented with premature adrenarche at the age of 6 years and menarche at the age of 12 years. On physical examination, her height was 154.5 cm and weight 50 kg, while she presented with acne, hirsutism, clitoromegaly, and normal blood pressure. Laboratory investigation revealed increased androgen levels and poor cortisol response to the ACTH stimulation test. From the family history, the mother was diagnosed with CAH at the age of 10 years and was under treatment with methylprednisolone. Previous molecular investigation of the CYP21A2 gene was negative. Due to the increased androstenedione levels in the index patient, the suspicion of 11βOH was raised, and she was investigated for 11-deoxycortisol, 11-deoxycorticosterone, and CYP11B1 gene pathogenic variants. The patient and her mother were found to be compound heterozygous for two novel variants of the CYP11B1 gene. Conclusion We present a case of CAH due to compound heterozygosity of two novel pathogenic variants of the CYP11B1 gene, emphasizing the importance of molecular investigation in order to confirm clinical diagnosis and allow proper genetic counseling of the family.

Preterm birth is associated with immaturity of several crucial physiological functions notably those prevailing in the lung and kidney. Recently, a steroid secretion deficiency was identified in very preterm neonates, associated with a partial yet transient deficiency in 11β-hydroxylase activity, sustaining cortisol synthesis. However, the P450c11β enzyme is expressed in preterm adrenal glands, we hypothesized an inhibition of cortisol production by adrenomedullin (ADM), a peptide highly produced in neonates and whose effect on steroidogenesis remains poorly known. We studied the effects of ADM on three models: 104 cord-blood samples of the PREMALDO neonate cohort, genetically targeted mice overexpressing ADM, and two human adrenocortical cell lines (H295R and HAC15 cells). Mid-regional-proADM (MR-proADM) quantification in cord-blood samples showed strong negative correlation with gestational age (P = 0.0004), cortisol production (P < 0.0001), and 11β-hydroxylase activity index (P < 0.0001). Mean MR-proADM was higher in very preterm than in term neonates (1.12 vs 0.60 nmol/L, P < 0.0001). ADM-overexpression mice revealed a lower 11β-hydroxylase activity index (P < 0.05). Otherwise, aldosterone levels measured by LC-MS/MS were higher in ADM-overexpression mice (0.83 vs 0.46 ng/mL, P < 0.05). More importantly, the negative relationship between adrenal ADM expression and aldosterone production found in control was lacking in the ADM-overexpression mice. Finally, LC-MS/MS and gene expression studies on H295R and HAC15 cells revealed an ADM-induced inhibition of both cortisol secretion in cell supernatants and CYP11B1 expression. Collectively, our results converge toward an inhibitory effect of ADM on glucocorticoid synthesis in humans and should be considered to explain the steroid secretion deficiency observed at birth in premature newborns.

The widespread environmental contaminant di-n-butyl phthalate (DBP) has been linked with reduced testosterone levels and adverse reproductive health outcomes in men. However, the underlying mechanisms of these anti-androgenic effects and the potential effects on other classes of steroid hormones remain to be elucidated. Here, we conducted mechanistic studies in human adrenocortical H295R cells exposed to 1-500 µM of DBP or its metabolite, mono-n-butyl phthalate (MBP), for 48 h. Quantification of steroid hormones in the cell medium by liquid chromatography-mass spectrometry revealed that both phthalates significantly decreased testosterone, androstenedione, corticosterone, and progesterone levels, in particular after dibutyryl-cyclic-AMP stimulation of steroidogenesis. Western blot analysis of key steroidogenic proteins showed that DBP induced a dose-dependent decrease of CYP11A1 and HSD3β2 levels, while MBP only significantly decreased CYP17A1 levels, indicating that the compounds affect early steps of the steroidogenesis differently. Both DBP and MBP exposure also lead to a dose-related decrease in HSD17β3, the enzyme which catalyzes the final step in the testosterone biosynthesis pathway, although these effects were not statistically significant. Interestingly, DBP increased the cortisol concentration, which may be due to the non-significant CYP11B1 increase in DBP-exposed cells. In contrast, MBP decreased cortisol concentration. Moreover, the analysis of superoxide generation and quantification of the protein oxidation marker nitrotyrosine demonstrated that DBP induced oxidative stress in H295R cells while MBP reduced protein nitrotyrosine levels. These findings confirm the anti-androgenic effects of DBP and MBP and reveal several differences in their toxicological mechanisms, with possible implications for future research on phthalate toxicity.

Primary aldosteronism (PA), including aldosterone and cortisol producing adenoma (A/CPA), is the most common trigger of secondary hypertension. The prevalence of A/CPA may be higher than what we could recognize previously with similar studies, but only a few relevant immunohistochemical reports have confirmed this information. Collecting more clinical features and immunohistochemistry data may help us to understand A/CPA, which is very important for avoiding misdiagnosis and improving outcomes in patients with A/CPA.Both individuals included in this study had hypertension for10 years. Computed tomography scans revealed the presence of adrenal nodules 1 year ago in patient A and 10 months ago in patient B (based on the date of the final version of this report). The relevant clinical features support PA accompanied by subclinical Cushing syndrome.Aldosterone and cortisol producing adenoma.The adrenal adenoma on the affected side was removed and pathological examination and immunohistochemistry were performed. Both the patients received short-term hydrocortisone treatment.The blood pressure of both patients improved after surgery. Cytochrome P450 (CYP)11B1, CYP11B2, parathyroid hormone receptor 1 (PTH1R), calcium-sensing receptor (CaSR), and vitamin D3 receptor (VD3R) were all positively expressed, but the histopathological features of the expression region differed.The occurrence of A/CPA may be related to calcium metabolism disorders. For A/CPA, the diversity in immunohistochemistry suggests many uncertainties regarding the pathogenesis of the disease. A/CPA should be considered in new clinical and pathological classifications of PA to gain more attention from the medical community.

Human cytochrome P450 11B1 (CYP11B1) generation of the major glucocorticoid cortisol requires two electrons delivered sequentially by the iron‑sulfur protein adrenodoxin. While the expected adrenodoxin binding site is on the opposite side of the heme and 15-20 Å away, evidence is provided that adrenodoxin allosterically impacts CYP11B1 ligand binding and catalysis. The presence of adrenodoxin both decreases the dissociation constant (K

Primary hyperaldosteronism (PAH) is the most frequent cause of secondary arterial hypertension. Most PAHs occur sporadically, but 5% of cases have a hereditary origin (familial PAH). Four forms of familial PAH have been described. Type I familial PAH is produced by a fusion of the CYP11B2 and CYP11B1 genes, in this way the synthesis of aldosterone becomes to be regulated by ACTH instead of by angiotensin II. In type II, III and IV familial PAH there is an increase in the transcription and expression of CYP11B2 responsible for aldosterone synthesis due to a germinal mutation in CLCN2, KCNJ5 and CACNA1H, respectively. On the other hand, somatic mutations have been identified in 50% of sporadic PAHs, with gain-of-function mutations at the level of KCNJ5, ATP1A1, ATP2B3 and CACNA1D being the most common. This review provides a detailed description of the different forms of familial PAH and the molecular profile of patients with sporadic PAH.

Context Syndromes of severe insulin resistance (SIR) include insulin receptoropathy, in which all signaling downstream of the insulin receptor is lost, and lipodystrophy, in which some signaling pathways are impaired and others preserved. Women with SIR commonly have ovarian hyperandrogenemia; adrenal-derived 11-oxygenated androgens, produced by CYP11B1, have not been studied. Objective We aimed to evaluate classic pathway androgens (androstenedione, testosterone) and 11-oxygenated androgens in women with SIR and hyperandrogenemia, and to elucidate the role of insulin receptor signaling for 11-oxygenated androgen production by comparing lipodystrophy and receptoropathy. Methods Steroid hormones were quantified using LC-MS/MS in a cross-sectional study of 18 women with hyperandrogenemia and SIR (11 lipodystrophy, 7 receptoropathy) and 23 controls. To assess ovarian vs adrenal origin, steroids were compared in receptoropathy patients with (Ovary+) vs without (Ovary-) ovarian function. Results Compared with controls, classic androgens were elevated in both lipodystrophy and receptoropathy, and 11-oxygenated androgens were increased in lipodystrophy (2.9-fold higher 11β-hydroxyandrostenedione (11OHA4), 2.4-fold higher 11-ketoandrostenedione (11KA4), 3.6-fold higher 11-ketotestosterone (11KT); P Conclusions 11-Oxygenated androgens are elevated in lipodystrophy but not receptoropathy. In SIR, insulin receptor signaling is necessary for adrenal hyperandrogenemia but not ovarian hyperandrogenemia; excess classic androgens are derived from the ovaries. Insulin receptor signaling increases adrenal 19-carbon steroid production, which may have implications for more common disorders of mild IR.

Background While previous studies indicate that the zonae reticularis (ZR) and glomerulosa (ZG) diminish with aging, little is known about age-related transformations of the zona fasciculata (ZF). Objectives To investigate the morphological and functional changes of the adrenal cortex across adulthood, with emphasis on (i) the understudied ZF and (ii) sexual dimorphisms. Methods We used immunohistochemistry to evaluate the expression of aldosterone synthase (CYP11B2), visinin-like protein 1 (VSNL1), 3β-hydroxysteroid dehydrogenase type II (HSD3B2), 11β-hydroxylase (CYP11B1), and cytochrome b5 type A (CYB5A) in adrenal glands from 60 adults (30 men), aged 18 to 86. Additionally, we employed mass spectrometry to quantify the morning serum concentrations of cortisol, 11-deoxycortisol (11dF), 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, and androstenedione in 149 pairs of age- and body mass index–matched men and women, age 21 to 95 years. Results The total cortical area was positively correlated with age (r = 0.34, P = 0.008). Both the total (VSNL1-positive) and functional ZG (CYP11B2-positive) areas declined with aging in men (r = −0.57 and −0.67, P < 0.01), but not in women. The CYB5A-positive area declined with age in both sexes (r = −0.76, P < 0.0001). In contrast, the estimated ZF area correlated positively with age in men (r = 0.59, P = 0.0006) and women (r = 0.49, P = 0.007), while CYP11B1-positive area remained unchanged across ages. Serum cortisol, corticosterone, and 11-deoxycorticosterone levels were stable across ages, while 11dF levels increased slightly with age (r = 0.16, P = 0.007). Conclusion Unlike the ZG and ZR, the ZF and the total adrenal cortex areas enlarge with aging. An abrupt decline of the ZG occurs with age in men only, possibly contributing to sexual dimorphism in cardiovascular risk.

Aldosterone is synthesized in the adrenal by the aldosterone synthase CYP11B2. Although the control of CYP11B2 expression is important to maintain the mineral homeostasis, its overexpression induced by the depolarization-induced calcium (Ca2+) signaling activation has been reported to increase the synthesis of aldosterone in primary aldosteronism (PA). The drug against PA focused on the suppression of CYP11B2 expression has not yet been developed, since the molecular mechanism of CYP11B2 transcriptional regulation activated via Ca2+ signaling remains unclear. To address the issue, we attempted to reveal the mechanism of the transcriptional regulation of CYP11B2 using chemical screening. We generated a cell line by inserting Nanoluc gene as a reporter into CYP11B2 locus in H295R adrenocortical cells using the CRSPR/Cas9 system, and established the high-throughput screening system using the cell line. We then identified 9 compounds that inhibited the CYP11B2 expression induced by potassium-mediated depolarization from the validated compound library (3399 compounds). Particularly, tacrolimus, an inhibitor of phosphatase calcineurin, strongly suppressed the CYP11B2 expression even at 10 nM. These results suggest that the system is effective in identifying drugs that suppress the depolarization-induced CYP11B2 expression. Our screening system may therefore be a useful tool for the development of novel medicines against PA.

Background Genetic factors are important risk factors to develop coronary heart disease (CHD). In this study, we mainly explored whether CYP11B1 mutations influence CHD risk among Chinese Han population. Methods Six variants were genotyped using Agena MassARRAY system from 509 CHD patients and 509 healthy controls. The correlations between CYP11B1 mutations and CHD risk were assessed using odds ratio (OR) and 95% confidence interval (95% CI) by logistic regression. The haplotype analysis and were ultifactor dimensionality reduction (MDR) were conducted. Results In the overall analysis, CYP11B1 polymorphisms were not correlated with CHD susceptibility. In the stratified analysis, we found that rs5283, rs6410, and rs4534 are significantly associated with susceptibility to CHD dependent on age and gender (p p rs4736312Ars5017238Crs5301Grs5283Trs6410Crs4534 haplotype of CYP11B1 reduce the susceptibility to CHD (p Conclusions We found that rs4534, rs6410 and rs5283 in CYP11B1 gene influence the susceptibility to CHD, which depend on age and gender.

Testicular adrenal rest tumor (TART) is one of the important complications that can cause infertility in male patients with congenital adrenal hyperplasia (CAH) and should therefore be diagnosed and treated at an early age. The factors that result in TART in CAH have not been completely understood. The aim of this study is to evaluate the genotype-phenotype correlation in CAH patients with TART.Method: Among 230 malepatients with CAH who were followed upwith regular scrotal ultrasonography in 11 different centers in Turkey, 40 patients who developed TARTand whose CAH diagnosis was confirmed by genetic testing were included in this study. Different approaches and methods were used for genotype analysis in this multicenter study. A few centers first screened the patients for the ten most common mutations in CYP21A2 and performed Sanger sequencing for the remaining regions only if these prior results were inconclusive while the majority of the departments adopted Sanger sequencing for the whole coding regions and exon-intron boundaries as the primary molecular diagnostic approach for patients with either CYP21A2 orCYP11B1 deficiency. The age of CAH diagnosis and TART diagnosis, type of CAH, and identified mutations were recorded.Results: TART was detected in 17.4% of the cohort [24 patients with salt-wasting (SW) type, four simple virilizing type, and one with nonclassical type with 21-hydroxylase (CYP21A2) deficiency and 11 patients with 11-beta hydroxylase (CYP11B1) deficiency]. The youngest patients with TART presenting with CYP11B1 and CYP21A2 deficiency were of 2 and 4 years, respectively. Eight different pathogenic variants in CYP21A2were identified. The most common genotypes were c.293-13C>G/c.293-13C>G (31%) followed by c.955C>T/ c.955C>T(27.6%) and c.1069C>T/c.1069C>T (17.2%). Seven different pathogenic variants were identified in CYP11B1. The most common mutation in CYP11B1 in our study was c.896T>C (p.Leu299Pro).Conclusion: We found that 83% TART patients were affected with SW typeCYP21A2 deficiency,and the frequent mutations detected were c.955C>T (p.Gln319Ter), c.293-13C>G in CYP21A2 and c.896T>C (p.Leu299Pro) inCYP11B1. Patients with CYP11B1 deficiency may develop TART at an earlier age. This study that examined the genotype-phenotype correlation in TART may benefit further investigations in larger series. WOS:000884450700002 Q3

Highlights • Prolonged MPRED treatment disrupts basal corticosterone ultradian rhythm. • Prolonged MPRED treatment decreases adrenal response to ACTH. • These effects are associated with decreased expression of steroidogenic proteins. • Prolonged MPRED treatment increases adrenal pro-inflammatory cytokine expression., Synthetic glucocorticoids are widely prescribed for the treatment of numerous inflammatory and autoimmune diseases and they can also affect the way the adrenal gland produces endogenous glucocorticoids. Indeed, patients undergoing synthetic glucocorticoid treatment can develop adrenal insufficiency, a condition characterised by reduced responsiveness of the adrenal to ACTH stimulation or stressors (e.g. surgical or inflammatory stress). To better elucidate the long-term effect of synthetic glucocorticoids treatment and withdrawal on adrenal function, we have investigated the long-term effects of prolonged treatment with methylprednisolone on HPA axis dynamics and on the adrenal steroidogenic pathway, both in basal conditions and in response to an inflammatory stress (lipopolysaccharide, LPS). We have found that 5-days treatment with methylprednisolone suppresses basal ACTH and corticosterone secretion, as well as corticosterone secretion in response to a high dose of ACTH, and down-regulates key genes in the adrenal steroidogenic pathway, including StAR, MRAP, CYP11a1 and CYP11b1. These effects were paralleled by changes in the adrenal expression of transcription factors regulating steroidogenic gene expression, as well as changes in the expression of adrenal clock genes. Importantly, 5 days after withdrawal of the treatment, ACTH levels are restored, yet basal levels of corticosterone, as well as most of the key steroidogenic genes and their regulators, remain down regulated. We also show that, although 5-days treatment with methylprednisolone reduces the corticosterone response to LPS, an increase in intra-adrenal pro-inflammatory cytokine gene expression was observed. Our data suggests that the steroidogenic pathway is directly affected by synthetic glucocorticoid treatment in the long-term, presumably via a mechanism involving activation of the glucocorticoid receptor. Furthermore, our data suggests a pro-inflammatory effect of synthetic glucocorticoids treatment in the adrenal gland.

Metabolic networks are complex cellular systems dependent on the interactions among, and regulation of, the enzymes in the network. Although there is great diversity of types of enzymes that make up metabolic networks, the models meant to understand the possible evolutionary outcomes following duplication neglect specifics about the enzyme, pathway context, and cellular constraints. To illuminate the mechanisms that shape the evolution of biochemical pathways, I functionally characterize the consequences of gene duplication of an enzyme family that performs multiple subsequent enzymatic reactions (a multistep enzyme) in the corticosteroid pathway in primates. The products of the corticosteroid pathway (aldosterone and cortisol) are steroid hormones that regulate metabolism and stress response in tetrapods. These steroid hormones are synthesized by a multistep enzyme Cytochrome P450 11B (CYP11B) that performs subsequent steps on different carbon atoms of the steroid derivatives. Through ancestral state reconstruction and in vitro characterization, I find that the primate ancestor of the CYP11B1 and CYP11B2 paralogs had moderate ability to synthesize both cortisol and aldosterone. Following duplication in Old World primates, the CYP11B1 homolog specialized on the production of cortisol, whereas its paralog, CYP11B2, maintained its ability to perform multiple subsequent steps as in the ancestral pathway. Unlike CYP11B1, CYP11B2 could not specialize on the production of aldosterone because it is constrained to perform earlier steps in the corticosteroid synthesis pathway to achieve the final product aldosterone. These results suggest that enzyme function, pathway context, along with tissue-specific regulation, both play a role in shaping potential outcomes of metabolic network elaboration.

Background Our study aims to summarize the clinical characteristics of rare types of congenital adrenal hyperplasia (CAH) other than 21-hydroxylase deficiency (21-OHD), and to explore the clinical applications of genetic analysis and liquid chromatography tandem-mass spectrometry (LC-MS/MS) in rare CAH. Methods We retrospectively analysed the clinical data of 5 rare cases of CAH admitted to our hospital and summarized their clinical manifestations, auxiliary examinations, diagnosis and mutational spectrum. Results After gene sequencing, complex heterozygous variants were detected in all patients (2 cases were lipoid congenital adrenal hyperplasia (LCAH), 11β-hydroxylase deficiency (11β-OHD), 3β-hydroxysteroid dehydrogenase deficiency (3β-HSD deficiency) and P450 oxidoreductase deficiency (PORD) each accounted for 1 case), which were consistent with their clinical manifestations. Among them, 4 novel variants were detected, including c.650 + 2 T > A of the StAR gene, c.1145 T > C (p. L382P) of the CYP11B1 gene, c.1622C > T (p. A541V) and c.1804C > T (p. Q602 *) of the POR gene. The LC-MS/MS results for steroid hormones in patients were also consistent with their genetic variants: 2 patients with LCAH showed a decrease in all steroid hormones; 11β-OHD patient showed a significant increase in 11-deoxycortisol and 11-deoxycorticosterone; patient with 3β-HSD deficiency showed a significant increase in DHEA; and PORD patient was mainly characterized by elevated 17OHP, progesterone and impaired synthesis of androgen levels. Conclusions The clinical manifestations and classification of CAH are complicated, and there are cases of missed diagnosis or misdiagnosis. It’s necessary to combine the analysis of clinical manifestations and auxiliary examinations for diagnosis; if necessary, LC-MS/MS analysis of steroid hormones or gene sequencing is recommended for confirming diagnosis and typing.

We performed genome-wide association study meta-analysis to identify genetic determinants of skeletal age (SA) deviating in multiple growth disorders. The joint meta-analysis (N = 4557) in two multiethnic cohorts of school-aged children identified one locus, CYP11B1 (expression confined to the adrenal gland), robustly associated with SA (rs6471570-A; β = 0.14; P = 6.2 × 10−12). rs6410 (a synonymous variant in the first exon of CYP11B1 in high LD with rs6471570), was prioritized for functional follow-up being second most significant and the one closest to the first intron-exon boundary. In 208 adrenal RNA-seq samples from GTEx, C-allele of rs6410 was associated with intron 3 retention (P = 8.11 × 10−40), exon 4 inclusion (P = 4.29 × 10−34), and decreased exon 3 and 5 splicing (P = 7.85 × 10−43), replicated using RT-PCR in 15 adrenal samples. As CYP11B1 encodes 11-β-hydroxylase, involved in adrenal glucocorticoid and mineralocorticoid biosynthesis, our findings highlight the role of adrenal steroidogenesis in SA in healthy children, suggesting alternative splicing as a likely underlying mechanism., Olja Grgic, Matthew Gazzara, and Alessandra Chesi et al. perform a genome-wide association study meta-analysis for skeletal age in two pediatric cohorts. They observe that variation in the adrenal gene, CYP11B1, impacts its alternative splicing, suggesting that adrenal steroidogenesis may contribute toward variation in skeletal age in otherwise healthy children.

OBJECTIVE Primary aldosteronism (PA) shows histological heterogeneity and clinical variability, including the coexistence of hypercortisolemia. Immunohistochemical analyses of steroidogenic enzymes in adrenal tissues have provided new insights into the pathogenesis of PA. However, a comprehensive analysis of the association between enzyme expression and clinical characteristics of PA has rarely been conducted. We aimed to investigate the correlation between clinical characteristics and steroidogenic enzyme expression in PA. DESIGN A retrospective case-control study. PATIENTS Consecutive patients who underwent unilateral adrenalectomy for PA (n = 180). Patients with adrenal Cushing's syndrome (CS) (n = 29) and nonfunctioning adenoma (n = 6) as comparator groups. MEASUREMENTS A tissue microarray of adrenal adenomas was constructed and immunostained for CYP11B1, CYP11B2 and CYP17A1. The expression of the three enzymes was compared between PA and other adrenal diseases and between PA with and without mild autonomous cortisol excess (MACE). RESULTS Adrenal adenomas in PA showed lower CYP11B1, higher CYP11B2 and lower CYP17A1 expression than those in adrenal CS (p

Context Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with limited treatment options. Theranostic approaches with adrenal specific radiotracers hold promise for improved diagnostics and treatment. Objective Here, we report a new theranostic approach to advanced ACC applying (R)-1-[1-(4-[123I]iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinyl amide ([123I]IMAZA) for diagnostic imaging and [131I]IMAZA for radionuclide therapy. Methods Sixty-nine patients with nonresectable, metastatic ACCs were screened using a diagnostic [123I]IMAZA scan. Patients with significant uptake in all tumoral lesions were offered treatment with [131I]IMAZA. Tumor response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST version 1.1), and adverse effects were assessed by Common Toxicity Criteria (version 5.0). Results After screening, 13 patients were treated with a median of 25.7 GBq [131I]IMAZA (range 18.1-30.7 GBq). Five individuals received a second treatment course. Best response was a decrease in the RECIST target lesions of –26% in 2 patients. Five patients with disease stabilization experienced a median progression-free survival of 14.3 months (range 8.3-21.9). Median overall survival in all patients was 14.1 months (4.0-56.5) after therapy. Treatment was well tolerated, in other words no severe toxicities (CTCAE grade ≥3) were observed. Conclusion In patients with advanced ACC refractory to standard therapeutic regimens, [131I]IMAZA treatment was associated with disease stabilization and nonsignificant tumor size reduction in a significant patient fraction and only limited toxicities. High [131I]IMAZA-uptake in tumor lesions was observed in 38.5% of patients with advanced ACC, rendering [131I] IMAZA a potential treatment option in a limited, well-defined patient fraction. Further clinical trials will be necessary to evaluate the full potential of this novel theranostic approach.

Objective Primary aldosteronism (PA) is the most common form of secondary and curable hypertension. Different germline and somatic mutations are found in aldosterone-producing adenoma (APA) and familial forms of the disease, while the causes of bilateral adrenal hyperplasia (BAH) remain largely unknown. Adrenalectomy is the recommended treatment for patients with APA; however, 6% of patients are not cured and show persistent PA after surgery suggesting BAH. The objective of this study was to analyze clinical data of patients with APA without biochemical success after adrenalectomy as well as the histological and genetic characteristics of their adrenal glands. Design and methods Clinical data of 12 patients with partial and absent biochemical cure were compared to those from 39 PA patients with hormonal cure after surgery. Histological, morphological, and genetic characterization of the adrenals was carried out by CYP11B2 and CYP11B1 immunostaining and by CYP11B2-guided NGS. Results Patients with absent hormonal cure displayed a longer duration of arterial hypertension and lower lateralization index of aldosterone production. In ten patients, APAs expressing CYP11B2 were identified. No difference in histological and morphological characteristics was observed between patients with or without a hormonal cure. Somatic mutations in APA driver genes were identified in all CYP11B2 positive APAs; CACNA1D mutations were the most frequent genetic abnormality. Conclusions Patients with partial and absent biochemical cure were diagnosed later and exhibited a lower lateralization index of aldosterone production, suggesting asymmetric aldosterone production in the context of BAH. Somatic mutations in adrenal glands from those patients indicate common mechanisms underlying BAH and APA.

IntroductionAdrenocortical hyperplasia and adrenal rest tumor (ART) formation are common in congenital adrenal hyperplasia (CAH). Although driven by excessive corticotropin, much is unknown regarding the morphology and transformation of these tissues. Our study objective was to characterize CAH-affected adrenals and ART and compare with control adrenal and gonadal tissues.Patients/MethodsCAH adrenals, ART and control tissues were analyzed by histology, immunohistochemistry, and transcriptome sequencing. We investigated protein expression of the ACTH receptor (MC2R), steroidogenic (CYP11B2, CYP11B1, CYB5A) and immune (CD20, CD3, CD68) biomarkers, and delta-like 1 homolog (DLK1), a membrane bound protein broadly expressed in fetal and many endocrine cells. RNA was isolated and gene expression was analyzed by RNA sequencing (RNA-seq) followed by principle component, and unsupervised clustering analyses.ResultsBased on immunohistochemistry, CAH adrenals and ART demonstrated increased zona reticularis (ZR)-like CYB5A expression, compared to CYP11B1, and CYP11B2, markers of zona fasciculata and zona glomerulosa respectively. CYP11B2 was mostly absent in CAH adrenals and absent in ART. DLK1 was present in CAH adrenal, ART, and also control adrenal and testis, but was absent in control ovary. Increased expression of adrenocortical marker MC2R, was observed in CAH adrenals compared to control adrenal. Unlike control tissues, significant nodular lymphocytic infiltration was observed in CAH adrenals and ART, with CD20 (B-cell), CD3 (T-cell) and CD68 (macrophage/monocyte) markers of inflammation. RNA-seq data revealed co-expression of adrenal MC2R, and testis-specific INSL3, HSD17B3 in testicular ART indicating the presence of both gonadal and adrenal features, and high expression of DLK1 in ART, CAH adrenals and control adrenal. Principal component analysis indicated that the ART transcriptome was more similar to CAH adrenals and least similar to control testis tissue.ConclusionsCAH-affected adrenal glands and ART have similar expression profiles and morphology, demonstrating increased CYB5A with ZR characteristics and lymphocytic infiltration, suggesting a common origin that is similarly affected by the abnormal hormonal milieu. Immune system modulators may play a role in tumor formation of CAH.

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.

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.

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 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.

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.

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.

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.

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.

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.

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

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.