The circadian rhythm of glucocorticoids has long been recognised within the last 75 years. Since the beginning, researchers have sought to identify basic mechanisms underlying the origin and emergence of the corticosteroid circadian rhythmicity among mammals. Accordingly, Young, Hall and Rosbash, laureates of the 2017 Nobel Prize in Physiology or Medicine, as well as Takahashi’s group among others, have characterised the molecular cogwheels of the circadian system, describing interlocking transcription/translation feedback loops essential for normal circadian rhythms. Plasma glucocorticoid circadian variation depends on the expression of intrinsic clock genes within the anatomic components of the hypothalamic–pituitary–adrenal axis, which are organised in a hierarchical manner. This review presents a general overview of the glucocorticoid circadian clock mechanisms, highlighting the ontogeny of the pituitary–adrenal axis diurnal rhythmicity as well as the involvement of circadian rhythm abnormalities in the physiopathology and diagnosis of Cushing’s disease.
Ayrton Custodio Moreira, Sonir Rauber Antonini, and Margaret de Castro
Silvia Liliana Ruiz Roa, Paula Conde Lamparelli Elias, Margaret Castro, and Ayrton Custodio Moreira
Cortisol awakening response (CAR) is a rapid increase of cortisol levels within 30–45 min after awakening.
This study evaluates CAR compared with cortisol circadian rhythm in active and in remission Cushing's disease (CD).
Materials and methods
We evaluated healthy controls (HC, n=19), obese (OB, n=10), in remission (n=08), and active CD patients (n=10). Salivary free cortisol (SF) was determined at 0800, 1100, 1700, 2000, and 2300 h on the first day. CAR was obtained the next morning immediately upon awakening and at 15, 30, 45, and 60-min post-wake up.
We observed differences in SF levels throughout the day in HC, OB, and in remission CD (ANOVA P=0.0001) but not in active CD (P=0.2). We demonstrated SF increment after awakening in HC, OB, and in remission CD (ANOVA P=0.007), with no effect of time on SF in active CD. The relative increment of SF obtained at the peak after awakening (CARi%) in the active CD (67±57%) was lower than in HC (154±107%), OB (240±188%), and in remission CD (186±184%) patients (P=0.009). There was a negative correlation between the SF at awakening and the CARi% in HC (r=−0.8), OB (r=−0.78), and in remission CD (r=−0.74) but not in active CD (r=−0.35; P=0.31).
This study originally described a blunted CAR in active CD in contrast to its presence in HC, OB, and in remission CD. This subtle dysfunction of the hypothalamus–pituitary–adrenal axis may represent a distinct and additional physiopathological phenomenon superimposing the dysregulated cortisol circadian rhythm in this disease.
Ana Carolina Bueno, Aniette R Espiñeira, Fábio L Fernandes-Rosa, Roberto Molina de Souza, Margaret de Castro, Ayrton Custódio Moreira, Heloísa Bettiol, Marco Antonio Barbieri, and Sonir R Antonini
To assess whether the −11391G>A polymorphism in the regulatory region of the adiponectin gene (ADIPOQ) is associated with birth size, postnatal growth, adiponectinemia, and cardiometabolic risk in adult life.
Case–control study nested within a prospective cohort of 2063 community subjects born in 1978/1979 and followed since birth to date.
ADIPOQ −11391G>A genotype–phenotype associations were evaluated in 116 subjects born large for gestational age (LGA) and 392 gender-matched controls at birth (birth size), at 8–10 years (catch-down growth), and at 23–25 years of age (cardiometabolic profile).
The −11391A variant allele frequency was higher in LGA subjects (P=0.04). AA genotype was associated with augmented probability of being born LGA (odds ratio=4.14; 95% confidence interval: 1.16–16.7; P=0.03). This polymorphism was associated neither with body composition nor with postnatal growth pattern. At the age of 23–25 years, the −11391A variant allele was associated with higher serum adiponectin levels (GG: 10.7±6.2 versus GA: 12.2±6.5 versus AA: 14.2±6.8 μg/ml; P<0.01). Subjects born LGA presented higher body mass index (BMI; P=0.01), abdominal circumference (P=0.04), blood pressure (P=0.04), and homeostasis assessment model for insulin resistance (P=0.01) than adequate for gestational age. Symmetry at birth did not influence these variables. The occurrence of catch-down of weight was associated with lower BMI and abdominal circumference (P<0.001) at 23–25 years.
The −11391A ADIPOQ gene variant was associated with increased chance of being born LGA and with higher adiponectin levels in early adult life.
Cristhianna Viesti Advincula Collares, Jose Antunes-Rodrigues, Ayrton Custodio Moreira, Suzana Nesi Franca, Luiz Alberto Pereira, Maria Marta Sarquis Soares, Jorge Elias Junior, Adrian J Clark, Margaret de Castro, and Lucila Leico Kagohara Elias
ACTH resistance syndromes are rare, autosomal, and genetically heterogeneous diseases that include familial glucocorticoid deficiency (FGD) and triple A syndrome. FGD has been shown to segregate with mutations in the gene coding for ACTH receptor (MC2R) or melanocortin 2 receptor accessory protein (MRAP), whereas mutations in the triple A syndrome (AAAS, Allgrove syndrome) gene have been found in segregation with triple A syndrome. We describe the clinical findings and molecular analysis of MC2R, MRAP, and AAAS genes in five Brazilian patients with ACTH resistance syndrome.
Design and methods
Genomic DNA from patients and their unaffected relatives was extracted from peripheral blood leucocytes and amplified by PCR, followed by automated sequencing. Functional analysis was carried out using Y6 cells expressing wild-type and mutant MC2R.
All five patients showed low cortisol and elevated plasma ACTH levels. One patient had achalasia and alacrima, besides the symptoms of adrenal insufficiency. The molecular analysis of FGD patients revealed a novel p.Gly116Val mutation in the MC2R gene in one patient and p.Met1Ile mutation in the MRAP gene in another patient. Expression of p.Gly116Val MC2R mutant in Y6 cells revealed that this variant failed to stimulate cAMP production. The analysis of the AAAS gene in the patient with triple A syndrome showed a novel g.782_783delTG deletion. The molecular analysis of DNA from other two patients showed no mutation in MC2R, MRAP, or AAAS gene.
In conclusion, the molecular basis of ACTH resistance syndrome is heterogeneous, segregating with genes coding for proteins involved with ACTH receptor signaling/expression or adrenal gland development and other unknown genes.
Deison Soares de Lima, Clarissa Silva Martins, Beatriz Maria de Carvalho Paixao, Fernando Colbari Amaral, Leandro Machado Colli, Fabiano P Saggioro, Luciano Neder, Helio Rubens Machado, Anemari Ramos Dinarte dos Santos, Daniel G Pinheiro, Ayrton Custodio Moreira, Wilson Araújo Silva Jr, and Margaret Castro
Although the molecular pathogenesis of pituitary adenomas has been assessed by several different techniques, it still remains partially unclear. Ribosomal proteins (RPs) have been recently related to human tumorigenesis, but they have not yet been evaluated in pituitary tumorigenesis.
The aim of this study was to introduce serial analysis of gene expression (SAGE), a high-throughput method, in pituitary research in order to compare differential gene expression.
Two SAGE cDNA libraries were constructed, one using a pool of mRNA obtained from five GH-secreting pituitary tumors and another from three normal pituitaries. Genes differentially expressed between the libraries were further validated by real-time PCR in 22 GH-secreting pituitary tumors and in 15 normal pituitaries.
Computer-generated genomic analysis tools identified 13 722 and 14 993 exclusive genes in normal and adenoma libraries respectively. Both shared 6497 genes, 2188 were underexpressed and 4309 overexpressed in tumoral library. In adenoma library, 33 genes encoding RPs were underexpressed. Among these, RPSA, RPS3, RPS14, and RPS29 were validated by real-time PCR.
We report the first SAGE library from normal pituitary tissue and GH-secreting pituitary tumor, which provide quantitative assessment of cellular transcriptome. We also validated some downregulated genes encoding RPs. Altogether, the present data suggest that the underexpression of the studied RP genes possibly collaborates directly or indirectly with other genes to modify cell cycle arrest, DNA repair, and apoptosis, leading to an environment that might have a putative role in the tumorigenesis, introducing new perspectives for further studies on molecular genesis of somatotrophinomas.
Ana Carolina Bueno, Mônica F Stecchini, Junier Marrero-Gutiérrez, Candy Bellido More, Leticia Ferro Leal, Débora Cristiane Gomes, Daniel Ferreira de Lima Neto, Silvia Regina Brandalise, Izilda Aparecida Cardinalli, José Andres Yunes, Thais Junqueira, Carlos Alberto Scrideli, Carlos Augusto Fernandes Molina, Fernando Silva Ramalho, Silvio Tucci, Fernanda Borchers Coeli-Lacchini, Ayrton Custodio Moreira, Leandra Ramalho, Ricardo Zorzetto Nicoliello Vêncio, Margaret De Castro, and Sonir Roberto R Antonini
Pediatric adrenocortical tumors (pACT) display complex genomic backgrounds, lacking robust prognostic markers and targeted therapeutic options. Vitamin D3 receptor (VDR) promoter hypermethylation and underexpression were reported in adrenocortical carcinomas from adult patients. In this study, we aimed to investigate VDR expression levels and methylation status in pACT and their clinical and prognostic significance.
Retrospective cross-sectional study enrolling pediatric patients with ACT from two tertiary referral institutions.
We evaluated clinicopathological features, VDR mRNA (qPCR) and protein (immunohistochemistry) expression, and VDR-wide methylation of ACT samples from 108 pediatric patients. Fourteen pediatric and 32 fetal and postnatal normal adrenals were used as controls.
Unlike in pre- and post-natal normal adrenals, most pACT lacked nuclear VDR expression and had reduced mRNA levels, especially the carcinomas. Unsupervised analysis of VDR methylation data revealed two groups of pACT with distinct disease features and outcomes. Tumors with high VDR methylation presented lower mRNA levels, and the respective patients presented advanced disease and reduced disease-free and overall survival.
VDR has a role in normal adrenocortical development and homeostasis, which is impaired during tumorigenesis. VDR hypermethylation and underexpression may be both predictive and prognostic biomarkers for pACT.
Jose Italo Soares Mota, Rui Milton Patrício Silva-Júnior, Clarissa Silva Martins, Ana Carolina Bueno, Luiz Eduardo Wildemberg, Ximene Lima da Silva Antunes, Jorge Guilherme Okanobo Ozaki, Fernanda Borchers Coeli-Lacchini, Carlos Garcia-Peral, Antonio Edson Rocha Oliveira, Antônio Carlos Santos, Ayrton Custodio Moreira, Helio Rubens Machado, Marcelo Volpon dos Santos, Leandro M Colli, Monica R Gadelha, Sonir Roberto R Antonini, and Margaret de Castro
To evaluate how telomere length behaves in adamantinomtous craniopharyngioma (aCP) and if it contributes to the pathogenesis of aCPs with and without CTNNB1 mutations.
Retrospective cross-sectional study enrolling 42 aCP patients from 2 tertiary institutions.
Clinicopathological features were retrieved from the patient’s charts. Fresh frozen tumors were used for RNA and DNA analyses. Telomere length was evaluated by qPCR (T/S ratio). Somatic mutations in TERT promoter (TERTp) and CTNNB1 were detected by Sanger and/or whole-exome sequencing. We performed RNA-Seq to identify differentially expressed genes in aCPs presenting with shorter or longer telomere lengths.
Mutations in CTNNB1 were detected in 29 (69%) tumors. There was higher frequency of CTNNB1 mutations in aCPs from patients diagnosed under the age of 15 years (85% vs 15%; P = 0.04) and a trend to recurrent disease (76% vs 24%; P = 0.1). No mutation was detected in the TERTp region. The telomeres were shorter in CTNNB1-mutated aCPs (0.441, IQR: 0.297–0.597vs 0.607, IQR: 0.445–0.778; P = 0.04), but it was neither associated with clinicopathological features nor with recurrence. RNAseq identified a total of 387 differentially expressed genes, generating two clusters, being one enriched for short telomeres and CTNNB1-mutated aCPs.
mutations are more frequent in children and adolescents and appear to associate with progressive disease. CTNNB1-mutated aCPs have shorter telomeres, demonstrating a relationship between the Wnt/β-catenin pathway and telomere biology in the pathogenesis of aCPs.