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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 genetic background of feline disorders of sex development (DSDs) is poorly understood. We performed comprehensive cytogenetic, molecular, and histological studies of 17 cats with abnormal external genitalia, unusual behavior, or tricolor coats (atypical in males). The DSD phenotype of three cats was associated with sex chromosome abnormalities: X/Y translocation (38,XXSRY+), 37,X/38,XY mosaicism, and XX/XY leukocyte chimerism. The remaining 14 affected cats were classified as XY DSD (SRY-positive). In this group and 38 normal males, we analyzed a priori selected candidate genes (SRY, TAC3, CYP11B1 and LHCGR). Only a previously reported nonpathogenic variant was found in SRY. Moreover, SRY gene copy number was determined, and three variants were observed: 6, 5 (modal), and 4 copies in a single DSD case. The known variants in TAC3 and CYP11B1, responsible for testicular hypoplasia, persistent primary dentition or congenital adrenal hyperplasia, were not found in the study group. Nine novel polymorphisms were identified in the LHCGR gene, one of which, a potentially regulatory indel variant in 5′UTR, was significantly associated (p = 0.0467) with XY DSD. Our report confirmed that abnormalities of sex chromosomes are important causes of feline DSDs. We also showed that the indel variant of LHCGR can be considered a promising marker associated with XY DSD phenotype.

Hyperaldosteronism is a common disease that is closely related to endocrine hypertension and other cardiovascular diseases. Cytochrome P450 11B2 (CYP11B2), an important enzyme in aldosterone (ALD) synthesis, is a promising target for the treatment of hyperaldosteronism. However, selective inhibitors targeting CYP11B2 are still lacking due to the high similarity with CYP11B1. In this study, atractylenolide-I (AT-I) was found to significantly reduce the production of ALD but had no effect on cortisol synthesis, which is catalyzed by CYP11B1. Chemical biology studies revealed that due to the presence of Ala320, AT-I is selectively bound to the catalytic pocket of CYP11B2, and the C8/C9 double bond of AT-I can be epoxidized, which then undergoes nucleophilic addition with the sulfhydryl group of Cys450 in CYP11B2. The covalent binding of AT-I disrupts the interaction between heme and CYP11B2 and inactivates CYP11B2, leading to the suppression of ALD synthesis; AT-I shows a significant therapeutic effect for improving hyperaldosteronism., Graphical abstract Atractylenolide-I (AT-I) is selectively oxidized in the presence of Ala320 of cytochrome P450 11B2 (CYP11B2), and then the epoxidized AT-I covalently binds to Cys450 of CYP11B2 via nucleophilic addition, inhibits ALD synthesis, and improves hyperaldosteronism.Image 1

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.

Inhibition of CYP11B1 is a promising therapy for severe diseases caused by excessive cortisol. Enantiomer discrimination provides clues to achieve selectivity that CYP11B1 and homologous CYP11B2 were selectively bound by

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.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by steroidogenic enzymes containing monogenetic defects. Most steroidogenic enzymes are cytochrome P450 groups that can be categorized as microsomal P450s, including 21-hydroxylase and 17α-hydroxylase/17,20 lyase, and mitochondrial P450s, including 11β-hydroxylase. It has been shown that ectopic administration of

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.

It still remains unclear how the functional organisation of the adrenal cortex arises. One aim of this study was to create a setup which allows for the establishment of a concentration gradient in vitro. This was achieved by a continuous flow of medium through the culture flask which caused differences in glucose and cortisol concentrations as well as in pH values between the sites of inflow and outflow of medium. Using real-time polymerase chain reaction, we found that a continuous supply of 1 ml medium per hour significantly increased the expression of MC2R, CYP11B1 and CYP17A1 genes of NCI-H295R cells in the distal area of the flask as compared with the proximal part. The expression of the AT1R showed a reverse regulation. The addition of dexamethasone to the medium led to an increase in gene expression of MC2R while AT1R was downregulated. Moreover, we detected a higher expression of CYP11B2 and a decreased expression of CYP11B1 when endothelial cell–conditioned medium (ECCM) was added to the inflow. Our experiments show that a directed medium delivery system creates different gradients and affects the functional differentiation of the NCI-H295R cells. Also, our results emphasise that products of endothelial cells have additional effects on the differentiation of the cultured adrenal cortical cells. Our results are in support that the regulation of the adrenal zonation is possible through different concentration gradients.

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.

Corticosterone is produced by the adrenal glands and also produced locally by other organs, such as the brain. Local levels of corticosterone in specific brain regions during development are not known. Here, we microdissected brain tissue and developed a novel liquid chromatography tandem mass spectrometry method (LC-MS/MS) to measure a panel of seven steroids (including 11-deoxycorticosterone (DOC), corticosterone, and 11-dehydrocorticosterone (DHC) in the blood, hippocampus (HPC), cerebral cortex (CC), and hypothalamus (HYP) of mice at postnatal day (PND) 5, 21, and 90. In a second cohort of mice, we measured the expression of three genes that code for steroidogenic enzymes that regulate corticosterone levels (Cyp11b1, Hsd11b1, and Hsd11b2) in the HPC, CC, and HYP. There were region-specific patterns of steroid levels across development, including higher corticosterone levels in the HPC and HYP than in the blood at PND5. In contrast, corticosterone levels were higher in the blood than in all brain regions at PND21 and PND90. Brain corticosterone levels were not positively correlated with blood corticosterone levels, and correlations across brain regions increased with age. Local corticosterone levels were best predicted by local DOC levels at PND5, but by local DHC levels at PND21 and PND90. Transcripts for the three enzymes were detectable in all samples (with highest expression of Hsd11b1) and showed region-specific changes with age. These data demonstrate that individual brain regions fine-tune local levels of corticosterone during early development and that coupling of glucocorticoid levels across regions increases with age.

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 Successful pregnancy with congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency is an extremely rare condition. Only two cases have been reported in the literature. Methods and results Described here is a 30-year-old woman diagnosed as a neonate with congenital adrenal hyperplasia related to 11-beta-hydroxylase deficiency classic type, who subsequently underwent clitoral resection and vaginoplasty. She was started on lifelong steroid therapy after surgery. She developed hypertension at 11 years of age and was on antihypertensive therapy from then on. In later life, she underwent division of vaginal scar tissue and perineal refashioning. She spontaneously conceived but her pregnancy was complicated by severe pre-eclampsia and delivery was required at 33 weeks of gestation by cesarean section. A healthy male infant was delivered. Conclusion Management of these women is similar to those with more common causes of congenital adrenal hyperplasia, with careful monitoring throughout pregnancy for complications such as gestational diabetes, gestational hypertension, and intrauterine growth restriction.

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.

Glucocorticoids (GCs) are critical modulators of the immune system. The hypothalamic-pituitary-adrenal (HPA) axis regulates circulating GC levels and is stimulated by endotoxins. Lymphoid organs also produce GCs; however, it is not known how lymphoid GC levels are regulated in response to endotoxins. We assessed whether an acute challenge of lipopolysaccharide (LPS) increases lymphoid levels of progesterone and GCs, and expression of steroidogenic enzymes and key HPA axis components (eg, corticotropin-releasing hormone [CRH], adrenocorticotropic hormone [ACTH]). We administered LPS (50 µg/kg intraperitoneally) or vehicle control to male and female C57BL/6J neonatal (postnatal day [PND] 5) and adult (PND90) mice and collected blood, bone marrow, thymus, and spleen 4 hours later. We measured progesterone, 11-deoxycorticosterone, corticosterone, and 11-dehydrocorticosterone via liquid chromatography-tandem mass spectrometry. We measured gene expression of key steroidogenic enzymes (Cyp11b1, Hsd11b1, and Hsd11b2) and HPA axis components (Crh, Crhr1, Pomc, and Mc2r) via quantitative polymerase chain reaction. At PND5, LPS induced greater increases in steroid levels in lymphoid organs than in blood. In contrast, at PND90, LPS induced greater increases in steroid levels in blood than in lymphoid organs. Steroidogenic enzyme transcripts were present in all lymphoid organs, and LPS altered steroidogenic enzyme expression predominantly in the spleen. Lastly, we detected transcripts of key HPA axis components in all lymphoid organs, and there was an effect of LPS in the spleen. Taken together, these data suggest that LPS regulates GC production by lymphoid organs, similar to its effects on the adrenal glands, and the effects of LPS might be mediated by local expression of CRH and ACTH.

of selective drug candidates for highly structural similar targets is a challenging task for researchers. The main objective of this study was to explore the selectivity modeling of pyridine and pyrimidine scaffold towards the highly homologous targets CYP11B1 and CYP11B2 enzymes by in silico (Molecular docking and QSAR) approaches. In this regard, a big dataset (n = 228) of CYP11B1 and CYP11B2 inhibitors were gathered and classified based on heterocyclic ring and the exhaustive analysis was carried out for pyridine and pyrimidinescaffolds. The LibDock algorithm was used to explore the binding pattern, screening, and identify the structural feature responsible for the selectivity of the ligands towards the studied targets. Finally, QSAR analysis was done to explore the correlation between various binding parameters and structural features responsible for the inhibitory activity and selectivity of the ligands in a quantitative way. The docking and QSAR analysis clearly revealed and distinguished the importance of structural features, functional groups attached for CYP11B2 and CYP11B1 selectivity for pyridine and pyrimidine analogs. Additionally, the docking analysis highlighted the differentiating amino acids residues for selectivity for ligands for each of the enzymes. The results obtained from this research work will be helpful in designing the selective CYP11B1/CYP11B2 inhibitors.

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

Objectives Congenital adrenal hyperplasia (CAH) is an autosomal recessive inherited disorder of steroidogenesis.11β-hydroxylase deficiency and 17α-hydroxylase deficiency are two forms of CAH caused by defects of CYP11B1 and CYP17A1 respectively. Case presentation Two rare intronic variants were identified in suspected CAH patients. Though not located at the classic splicing sites, these two variants perturbed splicing based on minigene assays. One variant, NM_000497.4: c.240-157T>G of CYP11B1 identified in subject 1, resulted in the retention of 136 intronic nucleotides. The other variant, NM_000102.4: c.754-6 A>G of CYP17A1 identified in subject 2, leading to the retention of 5 intronic nucleotides. Both variants resulted in out-of-frame alteration of the respective transcript. Conclusion Cryptic splicing variants in the intronic regions contribute to the genetic defects of CAH. Minigene assay is useful to confirm the splice altering effect and make a definitive molecular diagnosis.

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.

The impacts of androgens and glucocorticoids on spermatogenesis have intrigued scientists for decades. 11β-hydroxylase, encoded by cyp11c1, is the key enzyme involved in the synthesis of 11-ketotestosterone and cortisol, the major androgen and glucocorticoid in fish, respectively. In the present study, a Cyp11c1 antibody was produced. Western blot and immunohistochemistry showed that Cyp11c1 was predominantly expressed in the testicular Leydig cells and head kidney interrenal cells. A mutant line of cyp11c1 was established by CRISPR/Cas9. Homozygous mutation of cyp11c1 caused a sharp decrease of serum cortisol and 11-ketotestosterone, and a delay in spermatogenesis which could be rescued by exogenous 11-ketotestosterone or testosterone, but not cortisol treatment. Intriguingly, this spermatogenesis restored spontaneously, indicating compensatory effects of other androgenic steroids. In addition, loss of Cyp11c1 led to undersized testes with a smaller efferent duct and disordered spermatogenic cysts in adult males. However, a small amount of viable sperm was produced. Taken together, our results demonstrate that cyp11c1 is important for testicular development, especially for the initiation and proper progression of spermatogenesis. 11-ketotestosterone is the most efficient androgen in tilapia.

Unlike sex steroids, mineralocorticoids have attracted limited attention in ovarian physiology. Recent studies on primates have indicated possible local synthesis and action of mineralocorticoids in the ovary. Here, we examined developmental changes in the levels of mineralocorticoids and expression of genes encoding their biosynthetic enzymes and receptor in the bovine ovary. The follicles and corpora lutea (CL) were collected from F1 heifers. Expression levels of 21α-hydroxylase (CYP21A2), 11β-hydroxylase-1 (CYP11B1), and the mineralocorticoid receptor (NR3C2) in granulosa cells (GC), thecal layers (TL), and CL tissues were quantified by real-time PCR, whereas mineralocorticoids in the follicular fluid were measured by enzyme immunoassay (EIA). TL and GC expressed CYP21A2 and NR3C2, whereas CYP11B1 was expressed at very low or undetectable levels. The expression levels of these genes were not significantly different among small/large and healthy/atretic follicles but were higher in TL than in GC. CYP21A2 and NR3C2 were expressed in all CL stages with higher expression observed in the mid-stage. CYP11B1 expression was only apparent in the mid-stage CL. Aldosterone was detected in all follicles, and its concentration was not significantly different among the follicular groups. In paired large-healthy/atretic follicles, the concentration of deoxycorticosterone, a precursor of aldosterone, was approximately ten-fold higher than that of aldosterone and not significantly different between healthy and atretic follicles. In conclusion, the presence of mineralocorticoids and expression of NR3C2 in the bovine follicle together with the developmental change in the expression of CYP21A2, CYP11B1, and NR3C2 in the CL suggest possible endocrine/paracrine/autocrine roles of mineralocorticoids in the bovine ovary.

Congenital adrenal hyperplasia (CAH) is a group of inherited autosomal recessive disorders characterized by a defect in enzymes involved in biosynthesis of cortisol, aldosterone or both. We report two patients presented with recurrent haematuria with two different forms of CAH who presented late and reared as males. We describe the challenges posed on managing them and how the quality of life will be improved by offering hormonal and surgical remedies without changing or reassigning the gender to females.

Largemouth bass (Micropterus salmoides) is an economically important fish. It can spawn many times during a breeding season, and there are no obvious morphological characteristics to distinguish male and female juvenile fish. So far, little is known about the genes regulating their sexual development in this species. Here, we performed RNA sequencing (RNA-Seq) analysis of the testis, ovary, and somatic tissue to identify sex-related genes in the largemouth bass. A total of 51,672 unigenes were obtained via the transcriptome analysis, and 5900 differential expression genes (DEGs), including 3028 up-regulated and 2872 down-regulated DEGs, were obtained in the somatic tissue, testis, and ovary. DEGs were retrieved by making comparisons: somatic tissue vs testis (1733-up and 1382-down), testis vs ovary (841-up and 807-down), and ovary vs somatic tissue (454-up and 683-down). Finally, functional annotation identified 22 key sex-related DEGs, including 13 testis-biased DEGs (dmrt1, cyp11b1, sox9, spata4, spata22, spata17, fshr, fem-1a, wt1, daz1, amh, vasa, and piwi1) and 9 ovary-biased DEGs (foxl2, gdf9, zp3, sox3, cyp19a, bmp15, fem-1b, fig. la, and piwi2). This result was further confirmed by the tissue expression detection via RT-PCR and RT-qPCR. Protein-protein interacting (PPI) network analysis revealed that the testis-specific dmrt1 interacts directly with the testis-biased DEGs (cyp11b1 and spata4) and the ovary-biased DEGs (foxl2, gdf9, zp3, sox3, cyp19a, and bmp15), suggesting that the dmrt1 as a sex-determining gene can play a dual role through inducing the testis-biased DEGs and inhibiting the ovary-biased DEGs during the testicular development. Our present results provide useful molecular data for a better understanding of sexual development in the largemouth bass.

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

UV rays constitute an extremely important environmental factor known to operate adaptative mechanisms that maintain biological homeostasis in the skin, adrenal glands, and the brain. The skin is extremely vulnerable to UV rays. UV rays deform collagen, the main component of elastic fibers, decreasing its normal function, and ultimately reducing skin's elasticity. We confirmed that psychological stress occurring during the early stages of UVB-irradiation degraded collagen function by inhibiting production rather than the decomposition of collagen, thereby promoting skin aging. UV irradiation for 0-2 weeks increased the level of a stress factor, corticosterone (CORT). High-performance liquid chromatography and western blot analysis confirmed that the increase was caused by enhanced CYP11B1/2 levels during steroid synthesis in the adrenal gland. Precursor levels decreased significantly during the two weeks of UV irradiation. Skin collagen and collagen fibers reduced drastically during this time. Furthermore, the administration of osilodrostat, a USFDA-approved drug that selectively inhibits CYP11B1/2, preserved skin collagen. The mechanism underlying the reduction of CORT by osilodrostat confirmed that the amount of skin collagen could be preserved with treatment. In addition, upon suppression of the CORT receptor, the amount of collagen was controlled, and skin aging was suppressed by the hypothalamic-pituitary-adrenal axis. Therefore, this study confirmed an inverse relationship between adrenal CYP11B1/2 levels and collagen during the initial stages of UV irradiation of the skin. The findings of this study may be useful for developing new detection mechanisms for aging, following their further verification.

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.

Background Measurement of multiple steroids, 17 hydroxyprogesterone, 11 deoxycortisol, and 21 deoxycortisol, is required to discriminate between congenital adrenal hyperplasia due to 21 hydroxylase deficiency and that due to 11 beta hydroxylase deficiency. This work aims at the selection of the more appropriate, cost-effective method among either mass spectrometry or radioimmunoassay for the quantitation of the previous steroids. In this study, blood samples were collected from 31 patients that were newly diagnosed with congenital adrenal hyperplasia; 17 hydroxyprogesterone and 21 deoxycortisol were assayed using tandem mass spectrometry. Eleven deoxycortisol was assayed using 2 methods: radioimmunoassay and tandem mass spectrometry. Results Measuring 11 deoxycortisol using tandem mass spectrometry could significantly discriminate patients with 11 beta hydroxylase deficiency from those with 21 hydroxylase deficiency (p = 0.002), whereas radioimmunoassay failed (p = 0.095). Moreover, the former was highly predictive of 11 beta hydroxylase deficiency at a cutoff ≥ 11 ng/ml with 100% sensitivity and 92.3% specificity. Simultaneous measurement of 21 deoxycortisol and 11 deoxycortisol and their enrollment in an equation yielded an overall predictive accuracy 96.8% for diagnosis of CAH due to both enzymatic deficiencies. Conclusions Measurement of 11 deoxycortisol using mass spectrometric approach is mandated as a part of work up to differentiate types of congenital adrenal hyperplasia.