Catch-up growth and discontinuation of fludrocortisone treatment in aldosterone synthase deficiency

Background Aldosterone synthase deficiency (ASD) caused by mutations in the CYP11B2 gene is characterized by isolated mineralocorticoid deficiency. Data are scarce regarding clinical and biochemical outcomes of the disease in the follow-up. Objective Assessment of the growth and steroid profiles of patients with ASD at the time of diagnosis and after discontinuation of treatment. Design and method Children with clinical diagnosis of ASD were included in a multicenter study. Growth and treatment characteristics were recorded. Plasma adrenal steroids were measured using liquid chromatography-mass spectrometry. Genetic diagnosis was confirmed by CYP11B2 gene sequencing and in silico analyses. Results: Sixteen patients from 12 families were included (8 females; median age at presentation:3.1 months, range:0.4-8.1). The most common symptom was poor weight gain (56.3%).Median age of onset of fludrocortisone treatment was 3.6 months (0.9-8.3). Catch-up growth was achieved at median 2 months (0.5-14.5) after treatment. Fludrocortisone could be stopped in five patients at a median age of 6.0 years (2.2-7.6). Plasma steroid profiles revealed reduced aldosterone synthase activity both at diagnosis and after discontinuation of treatment compared to age-matched controls.We identified six novel (p.Y195H, c.1200 + 1G>A, p.F130L, p.E198del, c.1122-18G>A, p.I339_E343del) and four previously described CYP11B2 variants. The most common variant was p.T185I (40%). Conclusions Fludrocortisone treatment is associated with a rapid catch-up growth and control of electrolyte imbalances in ASD. Decreased mineralocorticoid requirement over time can be explained by the development of physiological adaptation mechanisms rather than improved aldosterone synthase activity. As complete biochemical remission cannot be achieved, a long-term surveillance of these patients is required.

Aldosterone Synthase Structure With Cushing Disease Drug LCI699 Highlights Avenues for Selective CYP11B Drug Design

Primary aldosteronism, the major form of secondary hypertension, develops due to excess steroid hormone aldosterone produced by aldosterone synthase, also known as cytochrome P450 11B2. CYP11B2 is 93% identical to cortisol-producing CYP11B1, which makes it difficult to design selective drugs. LCI699 (Osilodrostat, Isturisa) was initially developed as a CYP11B2 inhibitor but due to poor selectivity was recently repurposed as the first Food and Drug Administration-approved drug for CYP11B1-mediated Cushing disease. Thus, there is still no effective therapeutic option targeting CYP11B2 for primary aldosteronism. Using a structure/function approach, aspects of LCI699 interaction with CYP11B2 were examined to facilitate the design of more selective inhibitors. LCI699 effectively binds and inhibits CYP11B2. To determine the structural basis for this interaction, X-ray crystallography was used to solve the structure of CYP11B2 bound to LCI699. LCI699 binds in the active site with its imidazole nitrogen coordinating the heme iron. LCI699 binding was compared with that of its analog fadrozole to both CYP11B enzymes. Comparison with the CYP11B1 structure reveals distinct CYP11B active site architectures which can be exploited to directed drug design. Exploiting structural differences between the CYP11B enzymes will promote the design of therapeutics for the treatment of primary aldosteronism targeting CYP11B2 while reducing undesirable side effects due to off-target CYP11B1 inhibition.

Interaction Between Wnt/β-catenin and ACTH Signaling Pathways and Paracrine Regulation in Aldosterone Producing Adenoma

Primary aldosteronism (PA) is the most frequent form of secondary arterial hypertension and is caused in the majority of cases by an aldosterone producing adenoma (APA) or bilateral adrenal hyperplasia. Different somatic mutations have been identified in APA and in other aldosterone producing structures, which can be distinct within the same adrenal, suggesting multiple mechanisms underlying APA development. Also, APA show important cellular and molecular heterogeneity which may be due to interaction of different signaling pathways involved in adrenal cortex cell differentiation and function. The aim of this study was to investigate the role of Wnt/β-catenin and ACTH signaling as well as elements of paracrine regulation of aldosterone biosynthesis and vascularization in the development of APA and aldosterone producing cell clusters (APCC) and their relationship with intratumoral heterogeneity and mutational status. We performed immunohistochemistry and multiplex immunofluorescence (CYP11B2, CYP17A1, β-catenin, MC2R, pCREB, Tryptase, S100, CD34) multispectral image analysis on 11 adrenals with APA and one with micronodular hyperplasia from patients with PA. CYP11B2 (aldosterone synthase) IHC guided RT-qPCR was performed on RNA extracted from formalin-fixed paraffin-embedded tissues in 7 adrenals. Multiplex immunofluorescence revealed high abundance of tryptase positive mast cells and a dense vascular component in APA, which were independent of the mutational status. Within APA, mast cells were mainly localized in zones expressing CYP11B2, but not in areas expressing CYP17A1, and were rarely colocalized with nerve fibers, suggesting that their activity is not controlled by innervation. In cells expressing aldosterone synthase, β-catenin was activated, i.e. shows nuclear and/or cytoplasmic staining, features suggestive of a zona glomerulosa cell identity; MC2R was found at the cell membrane. Expression of MC2R mRNA was observed at different levels in APA, similar to expression of MRAP and VEGFA; MRAP2 was not detected. Within heterogeneous APA carrying KCNJ5 mutations, both MC2R and VEGFA expression was higher in areas expressing CYP11B2. Remarkably, this pattern was maintained in APCC, where cells show high CYP11B2 expression, together with activated β-catenin, independently of the mutation status. In addition, a high number of mast cells was detected around APCC, with a reorganization of the capillaries around the CYP11B2 positive cells. Our results suggest that aldosterone producing structures in adrenals with APA share common molecular characteristics and cellular environment, despite different mutation status. Mast cells appear to be closely associated with cells expressing aldosterone synthase, both in APA and APCC, and their role in regulating aldosterone biosynthesis in the context of somatic mutations in PA remains to be established.

DNA Methylation of the Angiotensinogen Gene, AGT, and the Aldosterone Synthase Gene, CYP11B2 in Cardiovascular Diseases

Angiotensinogen (AGT) and aldosterone play key roles in the regulation of blood pressure and are implicated in the pathogenesis of cardiovascular diseases. DNA methylation typically acts to repress gene transcription. The aldosterone synthase gene CYP11B2 is regulated by angiotensin II and potassium. DNA methylation negatively regulates AGT and CYP11B2 expression and dynamically changes in response to continuous promoter stimulation of each gene. High salt intake and excess circulating aldosterone cause DNA demethylation around the CCAAT-enhancer-binding-protein (CEBP) sites of the CYP11B2 promoter region, thereby converting the phenotype of AGT expression from an inactive to an active state in visceral adipose tissue and heart. A close association exists between low DNA methylation at CEBP-binding sites and increased AGT expression in salt-sensitive hypertensive rats. Salt-dependent hypertension may be partially affected by increased cardiac AGT expression. CpG dinucleotides in the CYP11B2 promoter are hypomethylated in aldosterone-producing adenomas. Methylation of recognition sequences of transcription factors, including CREB1, NGFIB (NR4A1), and NURR1 (NR4A2) diminish their DNA-binding activity. The methylated CpG-binding protein MECP2 interacts directly with the methylated CYP11B2 promoter. Low salt intake and angiotensin II infusion lead to upregulation of CYP11B2 expression and DNA hypomethylation in the adrenal gland. Treatment with the angiotensin II type 1 receptor antagonist decreases CYP11B2 expression and leads to DNA hypermethylation. A close association between low DNA methylation and increased CYP11B2 expression are seen in the hearts of patients with hypertrophic cardiomyopathy. These results indicate that epigenetic regulation of both AGT and CYP11B2 contribute to the pathogenesis of cardiovascular diseases.