Circulating adrenal and gonadal steroid hormones heterogeneity in active young males and the contribution of 11-oxy androgens.

The classical androgens, testosterone and dihydrotestosterone, together with dehydroepiandrosterone, the precusrsor to all androgens, are generally included in diagnostic steroid evaluations of androgen excess and deficiency disorders and monitored in androgen replacement and androgen suppressive therapies. The C11-oxy androgens also contribute to androgen excess disorders and are still often excluded from clinical and research-based steroids analysis. The contribution of the C11-oxy androgens to the androgen pool has not been considered in androgen deficiency. An exploratory investigation into circulating adrenal and gonadal steroid hormones in men was undertaken as neither the classical androgens nor the C11-oxy androgens have been evaluated in the context of concurrent measurement of all adrenal steroid hormones. Serum androgens, mineralocorticoids, glucocorticoids, progesterones and androgens were assessed in 70 healthy young men using ultra high performance supercritical fluid chromatography and tandem mass spectrometry. Testosterone, 24.5 nmol/L was the most prominent androgen detected in all participants while dihydrotestosterone, 1.23 nmol/L, was only detected in 25% of the participants. The 11-oxy androgens were present in most of the participants with 11-hydroxyandrostenedione, 3.37 nmol, in 98.5%, 11-ketoandrostenedione 0.764 in 77%, 11-hydroxytestosterone, 0.567 in 96% and 11-ketotestosterone: 0.440 in 63%. A third of the participants with normal testosterone and comparable 11-ketotestosterone, had significantly lower dehydroepiandrosterone (p < 0.001). In these males 11-hydroxyandrostenedione (p < 0.001), 11-ketoandrostenedione (p < 0.01) and 11-hydroxytestosterone (p < 0.006) were decreased. Glucocorticoids were also lower: cortisol (p < 0.001), corticosterone (p < 0.001), cortisone (p < 0.006) 11-dehydrocorticosterone (p < 0.001) as well as cortisol:cortisone (p < 0.001). The presence of dehydroepiandrosterone was associated with 16-hydroxyprogesterone (p < 0.001), which was also significantly lower. Adrenal and gonadal steroid analysis showed unexpected steroid heterogeneity in normal young men. Testosterone constitutes 78% of the circulating free androgens with the 11-oxy androgens abundantly present in all participants significantly contributing 22%. In addition, a subset of men were identified with low circulating dehydroepiandrosterone who showed altered adrenal steroids with decreased glucocorticoids and decreased C11-oxy androgens. Analysis of the classical and 11-oxy androgens with the additional measurement of dehydroepiandrosterone and 16-hydroxyprogesterone may allow better diagnostic accuracy in androgen excess or deficiency.

Steroidogenic cytochrome P450 enzymes as drug target.

Human cytochrome P450 (CYP) enzymes are composed of 57 individual enzymes that perform monooxygenase activities. They have diverse physiological roles in metabolizing xenobiotics and producing important endogenous compounds, such as steroid hormones and vitamins. At least seven CYP enzymes are involved in steroid biosynthesis. Steroidogenesis primarily occurs in the adrenal glands and gonads, connecting each reaction to substrates and products. Steroids are essential for maintaining life and significantly contribute to sexual differentiation and reproductive functions within the body. Disorders in steroid biosynthesis can frequently cause serious health problems and lead to the development of diseases, such as prostate cancer, breast cancer, and Cushing's syndrome. In this review, we provide current updated knowledge on the major CYP enzymes involved in the biosynthetic process of steroids, with respect to their enzymatic mechanisms and clinical implications for the development of new drug candidates.

Identification of sulfonylpyrimidines as novel selective aldosterone synthase (CYP11B2) inhibitors.

4-[(5-[2-Methyl-5-(methylsulfonyl)pentan-2-yl]sulfonylpyrimidin-4-yl)amino]benzonitrile 2 was identified as a novel potent aldosterone synthase inhibitor. Compound 2 was found to inhibit human CYP11B2 in the nanomolar range, and showed an aldosterone-lowering effect in a furosemide-treated cynomolgus monkey model. Although human CYP11B2 has the high homology sequence with human CYP11B1, compound 2 showed more than 80 times higher selectivity over human CYP11B1 in vitro.

Diagnostic and prognostic assessments of adrenocortical carcinomas by pathological features, immunohistochemical markers and reticular histochemistry staining.

BACKGROUND: Current diagnostic criteria of adrenocortical neoplasms are mostly based on morphology. The utility of immunohistochemistry (IHC) and histochemistry is limited. MATERIALS AND METHODS: To evaluate the diagnostic and prognostic utility of clinicopathological features, morphology, ancillary biomarkers, and reticular histochemistry in adrenocortical neoplasms. We examined 28 adrenocortical carcinomas (ACCs) and 50 adrenocortical adenomas (ACAs) obtained from pathology archives. Clinical data were retrieved from medical records. Two pathologists independently assessed hematoxylin and eosin-stained slides, employing modified Weiss criteria for all tumors and Lin-Weiss-Bisceglia criteria for oncocytic variants. Immunohistochemical markers (Calretinin, alpha-inhibin, MelanA, SF-1, Ki-67, PHH3, IGF-2, ß-catenin, P53, CYP11B1, CYP11B2, MLH1, MSH2, MSH6, PMS2, EPCAM) and Gomori's Silver histochemistry were applied. Statistical analysis utilized SPSS Statistics 26. RESULTS: ACCs exhibited larger tumor sizes (P<0.001) and symptomatic presentations (P = 0.031) compared to ACAs. Parameters of modified Weiss criteria and angioinvasion demonstrated diagnostic value for ACCs. Six immunohistochemical antibodies((MelanA, Ki-67, IGF-2, ß-catenin, P53 and CYP11B1) and reticulin framework alterations showed diagnostic value. Notably, Ki-67 and reticulin staining were most recommended. Evident reticulin staining was frequently present in ACCs (P<0.001). Ki-67 was significantly higher in ACCs (P<0.001). Twenty-one conventional and seven oncocytic entities showed different necrosis frequencies. Symptoms and Ki-67 index ≥ 30% were prognostic for ACCs, correlating with shorter survival. CONCLUSIONS: This study emphasizes the diagnostic value of reticulin framework alterations and a high Ki-67 index. Markers such as CYP11B1, IGF2, P53, ß-catenin and MelanA also contribute to the diagnosis of ACCs. Symptoms and Ki-67 index ≥ 30% predict shorter survival. These findings encourges the use of ancillary markers such as reticulin histochemistry and Ki-67 in the workup of evaluations of adrenocortical neoplasms.

Histopathological analysis of tumor microenvironment in adrenocortical carcinoma: Possible effects of in situ disorganized glucocorticoid production on tumor immunity.

Adrenocortical carcinoma (ACC) patients with glucocorticoid excess have been reported to be associated with decreased tumor-infiltrating immune cells, but the effects of in situ glucocorticoid production on tumor immunity have remained unknown. In addition, ACC was also known to harbor marked intra-tumoral heterogeneity of steroidogenesis or disorganized steroidogenesis. Therefore, in this study, we immune-profiled tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) and pivotal steroidogenic enzymes of glucocorticoid biosynthesis (CYP17A and CYP11B1) to explore the potential effects of in situ glucocorticoid production and intra-tumoral heterogeneity/disorganized steroidogenesis on tumor immunity of ACC. We also studied the correlations of the status of tumor immunity with that of angiogenesis and tumor grade to further explore the tumor tissue microenvironment of ACC. TILs (CD3, CD4, CD8, and FOXP3), TAMs (CD68 and CD163), key steroidogenic enzymes of glucocorticoid (CYP17A and CYP11B1), angiogenesis (CD31 and vasohibin-1 (VASH-1)), tumor grade (Ki-67 and Weiss score) were immunohistochemically evaluated in 34 ACCs. Increased CYP17A immunoreactivity in the whole tumor area was significantly positively correlated with FOXP3-positive TILs (p = 0.021) and negatively with CD4/CD3 ratio (p = 0.001). Increased CYP11B1 immunoreactivity in the whole tumor area was significantly positively correlated with CD8/CD3 (p = 0.039) and CD163/CD68 ratios (p = 0.006) and negatively with CD4-positive TILs (p = 0.036) and CD4/CD3 ratio (p = 0.001). There were also significant positive correlations between CYP17A and CD8 (r = 0.334, p < 0.001) and FOXP3-positive TILs (r = 0.414, p < 0.001), CD8/CD3 ratio (r = 0.421, p < 0.001), and CD68-positive TAMs (r = 0.298, p < 0.001) in randomly selected areas. Significant positive correlations were also detected between CYP11B1 and CD8/CD3 ratio (r = 0.276, p = 0.001) and negative ones detected between CYP11B1 and CD3- (r = -0.259, p = 0.002) and CD4-positive TILs (r = -0.312, p < 0.001) in those areas above. Increased micro-vessel density (MVD) -VASH-1 was significantly positively correlated with CD68- (p = 0.015) and CD163-positive TAMs (p = 0.009) and CD163/CD68 ratio and the high VASH-1 with CD163-positive TAMs (p = 0.042). Ki-67 labeling index was significantly positively correlated with MAD-VASH-1 (p = 0.006) and VASH-1 (p = 0.006) status. Results of our present study indicated that in situ glucocorticoid production did influence the status of tumor immunity in ACC. In particular, increased levels of CYP17A and CYP11B1, both involved in glucocorticoid producing immunoreactivity played different effects on tumor immunity, i.e., reflecting the involvement of intra-tumoral heterogeneity and disorganized steroidogenesis of ACC, which also did indicate the importance of in situ approaches when analyzing tumor immunity of ACC.

Cardiovascular risk assessments in patients with cortisol-producing adenoma: impact of clinical features and genetic characteristics.

The causes of adrenal Cushing's syndrome (CS) encompass a wide spectrum of adrenal cortisol proliferations that exhibit clinical and molecular heterogeneity. The aims of our study were to investigate whether clinical and molecular heterogeneity influences endothelial function and metabolic abnormalities in patients with cortisol-producing adenoma (CPA). We retrospectively enrolled 25 patients with CPA and 45 patients with essential hypertension (EH). All CPAs were studied by direct sequencing of PRKACA. Flow-mediated vasodilation (FMD), an index of vascular endothelial function, was significantly lower in CS and subclinical CS (SCS) groups than in the EH group. FMD impairment did not differ significantly between CS and SCS groups. No differences in FMD were seen between PRKACA mutant and wild-type groups. FMD correlated negatively with hemoglobin A1c (HbA1c) in both PRKACA mutant and wild-type groups, as well as in CS and SCS groups. After adrenalectomy, systolic blood pressure (SBP) and HbA1c decreased significantly from baseline in the CS group, and SBP and low-density lipoprotein cholesterol (LDL-C) decreased significantly from baseline in the SCS group. While SBP and LDL-C decreased significantly from baseline in patients with wild-type PRKACA, only HbA1c decreased from baseline in patients harboring PRKACA mutations. Our data showed that patients with CPA have impaired endothelial function compared with EH patients and suggest the need for strict monitoring of atherosclerosis, even in patients with SCS or without PRKACA mutation.

Clinical Presentation and Genetic Analysis of Neonatal 11ß-Hydroxylase Deficiency Induced by a Chimeric CYP11B2/CYP11B1 Gene.

In terms of prevalence, 11ß-hydroxylase deficiency (11ß-OHD), a common form of congenital adrenal hyperplasia, closely follows 21-hydroxylase deficiency. 11ß-OHD has been attributed to diminished enzymatic activity owing to CYP11B1 gene variants, mainly encompassing single nucleotide variations and insertions-deletions. The involvement of chimeric CYP11B2/CYP11B1 genes in 11ß-OHD has been rarely reported. We conducted a genetic investigation on a male infant with generalized pigmentation and abnormal steroid hormone levels. Whole-exome sequencing revealed a heterozygous variant in CYP11B1 inherited from the mother (NM_000497.4: c.1391_1393dup [p.Leu464dup]). Long-range polymerase chain reaction revealed an additional allele, a chimeric CYP11B2/CYP11B1 gene, inherited from the father. The current case report emphasizes the need to consider the occurrence of gene fusion variants in the diagnosis of neonatal or early infantile 11ß-OHD.

A common germline variant in CYP11B1 is associated with adverse clinical outcome of treatment with abiraterone or enzalutamide.

Extragonadal androgens play a pivotal role in prostate cancer disease progression on androgen receptor signaling inhibitors (ARSi), including abiraterone and enzalutamide. We aimed to investigate if germline variants in genes involved in extragonadal androgen synthesis contribute to resistance to ARSi and may predict clinical outcomes on ARSi. We included ARSi naive metastatic prostate cancer patients treated with abiraterone or enzalutamide and determined 18 germline variants in six genes involved in extragonadal androgen synthesis. Variants were tested in univariate and multivariable analysis for the relation with overall survival (OS) and time to progression (TTP) by Cox regression, and PSA response by logistic regression. A total of 275 patients were included. From the investigated genes CYP17A1, HSD3B1, CYP11B1, AKR1C3, SRD5A1 and SRD5A2, only rs4736349 in CYP11B1 in homozygous form (TT), present in 54 patients (20%), was related with a significantly worse OS (HR = 1.71, 95% CI 1.09 - 2.68, p = 0.019) and TTP (HR = 1.50, 95% CI 1.08 - 2.09, p = 0.016), and was related with a significantly less frequent PSA response (OR = 0.48, 95% CI 0.24 - 0.96, p = 0.038) on abiraterone or enzalutamide in a multivariable analysis. The frequent germline variant rs4736349 in CYP11B1 is, as homozygote, an independent negative prognostic factor for treatment with abiraterone or enzalutamide in ARSi naive metastatic prostate cancer patients. Our findings warrant prospective investigation of this potentially important predictive biomarker.

Case Report: A combination of chimeric CYP11B2/CYP11B1 and a novel p.Val68Gly CYP11B1 variant causing 11ß-Hydroxylase deficiency in a Chinese patient.

Introduction: 11ß-Hydroxylase deficiency (11ß-OHD, OMIM#202010) is the second most common form of congenital adrenal hyperplasia (CAH) caused by pathogenic variants in the CYP11B1 gene. Both single nucleotide variations (SNV)/small insertion and deletion and genomic rearrangements of CYP11B1 are important causes of 11ß-OHD. Among these variant types, pathogenic CYP11B2/CYP11B1 chimeras only contribute to a minority of cases. Heterozygote cases (chimera combined with SNV) are very rare, and genetic analysis of these cases can be challenging. Case presentation: We presented a suspected 11ß-OHD female patient with incomplete virilization, adrenal hyperplasia, and hypokalemia hypertension. Whole exome sequencing (WES) revealed that the patient carried both a chimeric CYP11B2/CYP11B1 and a novel missense variant, NM_000497.4: c.203T>G, p.Val68Gly (chr8:143961027) in CYP11B1, which were confirmed by CNVplex and Sanger sequencing, respectively. The patient's manifestations and genetic findings confirmed the diagnosis of 11ß-OHD, and oral dexamethasone was administered as a subsequent treatment. Conclusion: This report showed a rare CYP11B2/CYP11B1 chimera combined with a novel missense variant in a 11ß-OHD female patient. The result expands variant spectrum of CYP11B1 and suggests that both chimera and CYP11B1 variant screening should be performed simultaneously in suspected cases of 11ß-OHD. To our knowledge, this is the first report about CYP11B2/CYP11B1 chimera detected by WES analysis. WES combined with CNV analysis is an efficient method in the genetic diagnosis of this rare and complex disorder.

Generation of human steroidogenic cytochrome P450 enzymes for structural and functional characterization.

Six cytochrome P450 enzymes are involved in human steroidogenesis, converting cholesterol to sex steroids, mineralocorticoids, and glucocorticoids. While early work was accomplished with steroidogenic P450 orthologs from more accessible sources, knowledge of basic biochemistry through successful drug design have been greatly facilitated by recombinantly-expressed, highly purified human versions of these membrane proteins. Many membrane proteins are difficult to express and purify and are unstable. Membrane P450 expression in E. coli has been facilitated by modification and/or truncation of the membrane-interacting N-terminus, while metal-affinity resins and histidine-tagging greatly facilitates purification. However, substantial optimization is still frequently required to maintain protein stability. Over time, a generalized three-column purification scheme has been developed and tweaked to generate substantial quantities of fully active, highly purified human cytochrome P450 enzymes that have made possible the application of many structural, biochemical, and biophysical techniques to elucidate the mysteries of these critical human enzymes.

Pathogenicity of Congenital Adrenal Hyperplasia Induced by the p.P377L Mutation of CYP11B1.

CYP11B1 encodes an 11ß-hydroxylase that is involved in the catalysis of adrenal glucocorticoids and the production of cortisol. Mutations in CYP11B1 can result in congenital adrenal hyperplasia. We discovered a proband with a CYP11B1 gene mutation. Gene sequencing revealed a homozygous missense mutation of c.1130C > T in the 7th exon of the CYP11B1 gene that resulted in the change from Pro377 to leucine in the encoded protein. Based on the proband's clinical symptoms and the prognosis according to the database, this mutation may be harmful. However, the pathogenicity has not yet been reported. Thus, we created an expression vector for the mutation in vitro, transfected cells, observed the changes in gene expression, and determined its pathogenicity. To determine the pathogenicity of the CYP11B1 p.P377L mutation site through in vitro verification. The eukaryotic expression vector of the CYP11B1 mutation site was constructed in vitro, and the success of the construct was confirmed by sequencing. Fluorescence microscopy was used to determine the transfection effectiveness, GFP fluorescent tag labeling was used to detect changes in protein localization, and qRT‒PCR and Western blotting were used to detect CYP11B1 mRNA and protein expression. Sequencing revealed that the proband harbored a homozygous missense mutation of CYP11B1 (p.P377L). The expression of the protein decreased but the localization did not change when cells were transfected with the CYP11B1 mutation vector compared to the wild-type vector. The p.P377L mutation of CYP11B1 could affect protein expression and enzymatic activity and may be pathogenic.

Unequal crossing over between CYP11B2 and CYP11B1 causes 11 ß -hydroxylase deficiency in a consanguineous family.

Cytochrome P450 (CYP) family CYP11B2/CYP11B1 chimeric genes have been shown to arise from unequal crossing over of the genes encoding aldosterone synthase (CYP11B2) and 11ß-hydroxylase (CYP11B1) during meiosis. The activity deficiency or impaired activity of aldosterone synthase and 11ß-hydroxylase resulting from these chimeric genes are important reasons for 11ß-hydroxylase deficiency (11ß-OHD). Here，two patients with pseudoprecocious puberty and hypokalemia hypertension and three carriers in a consanguineous marriage family were studied. A single CYP11B2/CYP11B1 chimera consisting of the promoter and exons 1 through 5 of CYP11B2, exons 8 and 9 of CYP11B1, and a breakpoint consisting of part of exon 6 of CYP11B2 and part of exon 6, intron 6, and exon 7 of CYP11B1 were detected in the patients and carriers. At the breakpoint of the chimera, a c 0.1086 G > C ( p.Leu.362 =) synonymous mutation in exon 6 of CYP11B2, a c 0.1157 C＞G(p. A386V) missense mutation in exon 7 of CYP11B1, and an intronic mutation in intron 6 were detected. The allele model of the CYP11B2/CYP11B1 chimera demonstrated homozygosity and heterozygosity in the patients and the carriers, respectively. Molecular docking and enzymatic activity analyses indicated that the CYP11B2/CYP11B1 chimeric protein interacted with the catalytic substrate of aldosterone synthase and had similar enzymatic activity to aldosterone synthase. Our study indicated that deletion of CYP11B1 and CYP11B2 abolished the enzymatic activity of 11 ß-hydroxylase and aldosterone synthase; however, the compensation of the enzymatic activity of aldosterone synthase by the CYP11B2/CYP11B1 chimeric protein maintained normal aldosterone levels in vitro. All of the above findings explained the 11ß-OHD phenotypes of the proband and patients in the family.

Clinical and molecular characterization of 10 Chinese children with congenital adrenal hyperplasia due to 11beta-hydroxylase deficiency.

BACKGROUND: The clinical manifestations of nonclassical 11beta-hydroxylase deficiency are very similar to those of non-classical 21-hydroxylase deficiency. For this study, we investigated the relationship between the clinical and molecular features of congenital adrenal hyperplasia caused by 11beta-hydroxylase deficiency and reviewed the related literature, which are expected to provide assistance for the clinical diagnosis and analysis of congenital adrenal hyperplasia. METHODS: Clinical data for 10 Chinese patients diagnosed with congenital adrenal hyperplasia in our hospital from 2018 to 2022 were retrospectively analyzed. We examined the effects of gene mutations on protease activity and constructed three-dimensional structure prediction models of proteins. RESULTS: We describe 10 patients with 11beta-hydroxylase gene mutations (n = 5, 46,XY; n = 5, 46,XX), with 10 novel mutations were reported. Female patients received treatment at an early stage, with an average age of 2.08 ± 1.66 years, whereas male patients received treatment significantly later, at an average age of 9.77 ± 3.62 years. The most common CYP11B1 pathogenic variant in the Chinese population was found to be c.1360C > T. All mutations lead to spatial conformational changes that affect protein stability. CONCLUSIONS: Our study found that there was no significant correlation between each specific mutation and the severity of clinical manifestations. Different patients with the same gene pathogenic variant may have mild or severe clinical manifestations. The correlation between genotype and phenotype needs further study. Three-dimensional protein simulations may provide additional support for the physiopathological mechanism of genetic mutations.

Virtual screening and biological evaluation to identify pharmaceuticals potentially causing hypertension and hypokalemia by inhibiting steroid 11ß-hydroxylase.

Several drugs were found after their market approval to unexpectedly inhibit adrenal 11ß-hydroxylase (CYP11B1)-dependent cortisol synthesis. Known side-effects of CYP11B1 inhibition include hypertension and hypokalemia, due to a feedback activation of adrenal steroidogenesis, leading to supraphysiological concentrations of 11-deoxycortisol and 11-deoxycorticosterone that can activate the mineralocorticoid receptor. This results in potassium excretion and sodium and water retention, ultimately causing hypertension. With the risk known but usually not addressed in preclinical evaluation, this study aimed to identify drugs and drug candidates inhibiting CYP11B1. Two conceptually different virtual screening methods were combined, a pharmacophore based and an induced fit docking approach. Cell-free and cell-based CYP11B1 activity measurements revealed several inhibitors with IC50 values in the nanomolar range. Inhibitors include retinoic acid metabolism blocking agents (RAMBAs), azole antifungals, α2-adrenoceptor ligands, and a farnesyltransferase inhibitor. The active compounds share a nitrogen atom embedded in an aromatic ring system. Structure activity analysis identified the free electron pair of the nitrogen atom as a prerequisite for the drug-enzyme interaction, with its pKa value as an indicator of inhibitory potency. Another important parameter is drug lipophilicity, exemplified by etomidate. Changing its ethyl ester moiety to a more hydrophilic carboxylic acid group dramatically decreased the inhibitory potential, most likely due to less efficient cellular uptake. The presented work successfully combined different in silico and in vitro methods to identify several previously unknown CYP11B1 inhibitors. This workflow facilitates the identification of compounds that inhibit CYP11B1 and therefore pose a risk for inducing hypertension and hypokalemia.

Corrigendum: Challenges in the treatment of late-identified untreated congenital adrenal hyperplasia due to CYP11B1 deficiency: Lessons from a developing country.

[This corrects the article DOI: 10.3389/fendo.2022.1015973.].

Glucocorticoids are differentially synthesized along the murine and human respiratory tree.

BACKGROUND: Synthetic glucocorticoids (GC) are effective in the treatment of inflammatory diseases of the lung. However, long-term use leads to severe side effects. Endogenous GC can be synthesized locally, either de novo from cholesterol in a 11ß-hydroxylase (Cyp11b1)-dependent manner, or by reactivation from 11-dehydrocorticosterone/cortisone by 11ß-hydroxysteroid dehydrogenase 1 (Hsd11b1). We aimed to define the molecular pathways of endogenous GC synthesis along the respiratory tree to provide a basis for understanding how local GC synthesis contributes to tissue homeostasis. METHODS: Expression of steroidogenic enzymes in murine lung epithelium was analyzed by macroscopic and laser capture microdissection, followed by RT-qPCR. Flow cytometry analysis was performed to identify the cellular source of steroidogenic enzymes. Additionally, the induction of steroidogenic enzyme expression in the lung was analyzed after lipopolysaccharide (LPS) injection. mRNA and protein expression of steroidogenic enzymes was confirmed in human lung tissue by RT-qPCR and immunohistochemistry. Furthermore, GC synthesis was examined in ex vivo cultures of fresh tissue from mice and human lobectomy patients. RESULTS: We observed that the murine and human lung tissue differentially expresses synthesis pathway-determining enzymes along the respiratory tree. We detected Hsd11b1 expression in bronchial, alveolar, club and basal epithelial cells, whereas Cyp11b1 expression was detectable only in tracheal epithelial cells of mice. Accordingly, de novo synthesis of bioactive GC occurred in the large conducting airways, whereas reactivation occurred everywhere along the respiratory tree. Strikingly, Cyp11b1 but not Hsd11b1 expression was enhanced in the trachea upon LPS injection in mice. CONCLUSION: We report here the differential synthesis of bioactive GC along the murine and human respiratory tree. Thus, extra-adrenal de novo GC synthesis and reactivation may differentially contribute to the regulation of immunological and inflammatory processes in the lung.

Unexpectedly high mutation rate of cyp11b1 compared to cyp21a2 in randomly-selected turkish women: a large screening study.

PURPOSE: Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders resulting from enzyme deficiencies associated with steroidogenesis. The clinical presentation of non-classic CAH (NCAH) in females is often indistinguishable from other hyperandrogenic disorders like polycystic ovary syndrome (PCOS). The data on the prevalence of NCAH in unselected women in the literature is scanty. The research aimed to evaluate the prevalence of NCAH, carrier frequencies, and the correlation between clinical symptoms and genotype in Turkish women. METHODS: The study group comprised two hundred and seventy randomly-selected unrelated asymptomatic women of reproductive age (18-45). Subjects were recruited from female blood donors. All volunteers underwent clinical examination and hormone measurements. The protein-encoding exons and exon-intron boundaries of the CYP21A2, CYP11B1, HSD3ß2 and CYP21A2 promoter were sequenced by direct DNA sequencing. RESULTS: After genotyping, seven (2.2%) individuals were diagnosed with NCAH. The heterozygous carrier frequencies of CYP21A2, CYP21A2 promoter, CYP11B1, and HSD3ß2 genes with 34, 34, 41, and 1 pathologic mutation were determined at 12.6%, 12.6%, 15.2%, and 0.37% of volunteers, respectively. Gene-conversion (GC) frequencies between CYP21A2/CYP21A1P and CYP11B1/CYP11B2 were determined as 10.4% and 14.8%, respectively. CONCLUSION: Despite GC-derived higher mutation frequency determined in the CYP11B1 gene, the reason for the low frequency of NCAH due to 11OHD compared to 21OHD might be that gene-conversion arises with active CYP11B2 rather than an inactive pseudogene. HSD3ß1 exhibits high homology with HSD3ß2 located on the same chromosome; remarkably, it demonstrates low heterozygosity and no GC, most probably the outcome of a tissue-specific expression pattern.

Morphological Harbingers of ARMC5-Pathogenic Variant-Related Bilateral Macronodular Adrenocortical Disease.

Bilateral macronodular adrenocortical disease (BMAD) is a neoplastic disease associated with a high frequency of germline disease. Armadillo repeat containing 5 (ARMC5) pathogenic variants (PVs) have not been widely studied to determine the morphological and immunohistochemical characteristics of BMAD. We carried out a detailed morphologic review of 22 surgical specimens excised from patients with BMAD and compared them with PV of ARMC5 (PV + , n = 14) and those without (PV - , n = 8), and further comparing them with a control group of adrenals excised from patients with renal cancer (n = 11). No patients presented with a genetic syndrome related to BMAD. Overt Cushing's syndrome was present in 12/22 patients, 10 PV + and 2 PV - (p = 0.074). We also evaluated the expression of Ki-67, BCL-2, BAX, p53, CYP11B1, and ARMC5 protein. The pseudo-glandular and trabecular architectural patterns were strongly associated with the PV + group (both p < 0.001), as well as capsular extrusion (p < 0.001). There was no predictive value in the distinction of ARMC5 variants in Hsiao subtyping. ARMC5 diffuse cytoplasmic staining was observed in all 11 control samples. The ARMC5 expression was significantly lower in BMAD than in the control group (p < 0.001). In all the specimens, expression of BCL-2 was identified only in the medulla, and expression of BAX was observed in adrenocortical cells. CYP11B1 diffuse immunoexpression was identified in all the specimens of BMAD and in the fasciculata zone in the control group. The mitotic count and Ki-67 proliferation index was very low in all three groups (controls, PV + , and PV - BMAD). None of the specimens stained positive for the p53 protein. Although our series is limited, the presence of pseudo-glandular and/or trabecular patterns and capsular extrusion indicated the presence of pathogenic variants of ARMC5 in BMAD. The gland enlargement does not seem to be related to the increase of mitotic count or a higher proliferation index (Ki-67).

Immune escape of colorectal tumours via local LRH-1/Cyp11b1-mediated synthesis of immunosuppressive glucocorticoids.

Control of tumour development and growth by the immune system critically defines patient fate and survival. What regulates the escape of colorectal tumours from destruction by the immune system remains currently unclear. Here, we investigated the role of intestinal synthesis of glucocorticoids in the tumour development during an inflammation-induced mouse model of colorectal cancer. We demonstrate that the local synthesis of immunoregulatory glucocorticoids has dual roles in the regulation of intestinal inflammation and tumour development. In the inflammation phase, LRH-1/Nr5A2-regulated and Cyp11b1-mediated intestinal glucocorticoid synthesis prevents tumour development and growth. In established tumours, however, tumour-autonomous Cyp11b1-mediated glucocorticoid synthesis suppresses anti-tumour immune responses and promotes immune escape. Transplantation of glucocorticoid synthesis-proficient colorectal tumour organoids into immunocompetent recipient mice resulted in rapid tumour growth, whereas transplantation of Cyp11b1-deleted and glucocorticoid synthesis-deficient tumour organoids was characterized by reduced tumour growth and increased immune cell infiltration. In human colorectal tumours, high expression of steroidogenic enzymes correlated with the expression of other immune checkpoints and suppressive cytokines, and negatively correlated with overall patients' survival. Thus, LRH-1-regulated tumour-specific glucocorticoid synthesis contributes to tumour immune escape and represents a novel potential therapeutic target.

Molecular analysis of 12 Chinese patients with 11ß-hydroxylase deficiency and in vitro functional study of 20 CYP11B1 missense variants.

Steroid 11ß-hydroxylase deficiency (11ß-OHD) is a rare autosomal recessive disorder caused by pathogenic variants of CYP11B1 gene. This study aimed to perform molecular analysis of a Chinese 11ß-OHD series and in vitro functional study of twenty CYP11B1 missense variants. Twelve Chinese patients with clinical diagnosis of 11ß-OHD were included in the study to analyze their molecular etiology. Genomic DNA of patients was extracted to be sequenced all coding exons and intronic flanking sequences of CYP11B1. Fourteen missense variants found in 12 patients mentioned above along with 6 missense variants previously reported by our team were evaluated functionally. Amino acid substitutions were analyzed with computational program to determine their effects on the three-dimensional structure of CYP11B1 protein. Clinical characteristics and hormone levels at baseline of the 18 patients carrying 18 missense variants aforementioned were recorded to perform genotype-phenotype correlation. A total of 21 rare variants including 9 novel and 12 recurrent ones were identified in 12 patients, out of which 17 were missense, 2 were nonsense, 1 was a splice site variant, and 1 was a deletion-insertion variant. Results of in vitro functional study revealed that 3 out of 20 missense mutants (p.Leu3Pro, p.Gly267Ser, and p.Ala367Ser) had partial enzyme activity and the other 17 had little enzymatic activity. The impairment degree of enzymatic activity in vitro functional study was also reflected in the severity degree of interaction change between the wild-type/mutant-type amino acid and its adjacent amino acids in three-dimensional model. In conclusion, the addition of 9 novel variants expands the spectrum of CYP11B1 pathogenic variants. Our results demonstrate that twenty CYP11B1 variants lead to impaired 11ß-hydroxylase activity in vitro. Visualizing these variants in the three-dimensional model structure of CYP11B1 protein can provide a plausible explanation for the results measured in vitro.