Ayrton C. Moreira

MicroRNAs Differentially Expressed in ACTH-Secreting Pituitary Tumors

CONTEXT MicroRNAs (miRNAs) are small noncoding RNAs, functioning as antisense regulators of gene expression by targeting mRNA and contributing to cancer development and progression. More than 50% of miRNA genes are located in cancer-associated genomic regions or in fragile sites of the genome. OBJECTIVE The aim of the study was to analyze the differential expression of let-7a, miR-15a, miR-16, miR-21, miR-141, miR-143, miR-145, and miR-150 in corticotropinomas and normal pituitary tissue and verify whether their profile of expression correlates with tumor size or remission after treatment. MATERIAL AND METHODS ACTH-secreting pituitary tumor samples were obtained during transphenoidal surgery from patients with Cushing disease and normal pituitary tissues from autopsies. The relative expression of miRNAs was measured by real-time PCR using RNU44 and RNU49 as endogenous controls. Relative quantification of miRNA expression was calculated using the 2(-DeltaDeltaCt) method. RESULTS We found underexpression of miR-145 (2.0-fold; P = 0.04), miR-21 (2.4-fold; P = 0.004), miR-141 (2.6-fold; P = 0.02), let-7a (3.3-fold; P = 0.003), miR-150 (3.8-fold; P = 0.04), miR-15a (4.5-fold; P = 0.03), miR-16 (5.0-fold; P = 0.004), and miR-143 (6.4-fold; P = 0.004) in ACTH-secreting pituitary tumors when compared to normal pituitary tissues. There were no differences between miRNA expression and tumor size as well as miRNA expression and ratio of remission after surgery, except in patients presenting lower miR-141 expression who showed a better chance of remission. CONCLUSION Our results support the possibility that altered miRNA expression profile might be involved in corticotrophic tumorigenesis. However, the lack of knowledge about miRNA target genes postpones full understanding of the biological functions of down-regulated or up-regulated miRNAs in corticotropinomas.

Longitudinal evaluation of the development of salivary cortisol circadian rhythm in infancy

OBJECTIVE Although an outstanding characteristic of the adrenocortical function of children and adults is its circadian rhythm, little information is available about the age of appearance of such rhythm in infancy. The main obstacle has been the ethical difficulty in obtaining serial blood samples from healthy infants. We monitored the development of cortisol daily rhythm using non‐invasive salivary cortisol determination.

Salivary cortisol for screening of Cushing's syndrome in children

Cushings syndrome (CS) is characterized by changes in diurnal cortisol variation and total or partial resistance to cortisol suppression by dexamethasone (DEX). Diagnosing CS is a challenge especially in childhood and requires differentiation from primary obesity. The aim was verify the efficacy of salivary cortisol in differentiating primary obesity from CS in children.

Anxiety and salivary cortisol in symptomatic and nonsymptomatic panic patients and healthy volunteers performing simulated public speaking

Anxiety and salivary cortisol were measured in subjects performing simulated public speaking (SPS), a procedure that has been neurobiologically related to panic disorder. The subjects were divided into three groups: 18 symptomatic panic patients, 16 nonsymptomatic, drug-treated panic patients, and 17 healthy controls. In the experimental session, subjective anxiety (Visual Analogue Mood Scale) and the total score of the Bodily Symptom Scale (BSS) were higher in symptomatic patients than in controls, with nonsymptomatic patients in between. Measures of cortisol taken at home showed that the level was higher at 9:00 h than at 23:00 h in every group, indicating a normal circadian regulation of the hypothalamic-pituitary-adrenal (HPA) axis in panic patients. Also in every group, the level of cortisol was high at the beginning of the experimental session and decreased after 70 min. This fall parallels the decrease in anxiety and BSS ratings, and appears to reflect habituation of initial, anticipatory anxiety. Preparation and performance of speech raised anxiety and BSS scores to the initial levels, but failed to increase cortisol measured over 60 min, starting at the end of the speech. Therefore, SPS does not seem to activate the HPA axis, as reported in panic attacks.

Revised Guidelines for Neonatal Screening Programmes for Primary Congenital Hypothyroidism

Since the first guidelines for neonatal screening for congenital hypothyroidism (CH) were issued by ESPE in 1993 [1], there have been considerable advances in our understanding of CH and our appreciation of the various geographical and logistic difficulties involved. Therefore, an updating of the guidelines is overdue. Experience from countries where screening began in the late 1970s and early 1980s has indicated that treatment should be started no later than the first 2 weeks of life using a ‘high’ dosage regime of L-thyroxine (10–15 Ìg/kg/day). It has also been shown that the quality of long-term outcome is closely related to the quality of follow-up. In Eastern Europe, screening programmes for CH have either been started or will start soon in almost all countries, and although many programmes are operating satisfactorily, it is important to standardise screening procedures and management of suspected cases as much as possible in order to optimise outcome. A degree of uniformity throughout Europe would not only facilitate early detection and treatment of individual patients but give insight into the economic and epidemiological aspects of the screening programmes as well as the epidemiological aspect of CH.

The emergence of salivary cortisol circadian rhythm and its relationship to sleep activity in preterm infants

OBJECTIVE The circadian rhythm of cortisol is established at between 8 and 12 postnatal weeks in term infants. However, there is limited information about the effect of prematurity on this rhythm. We evaluated the emergence of the salivary cortisol circadian rhythm in premature infants and its relationship to the onset of sleep daily rhythm.

Salivary cortisol as a tool for physiological studies and diagnostic strategies

Salivary cortisol is an index of plasma free cortisol and is obtained by a noninvasive procedure. We have been using salivary cortisol as a tool for physiological and diagnostic studies, among them the emergence of circadian rhythm in preterm and term infants. The salivary cortisol circadian rhythm in term and premature infants was established between 8 and 12 postnatal weeks. In the preterm infants the emergence of circadian rhythm was parallel to the onset of sleep rhythm. We also studied the use of salivary cortisol for screening for Cushings syndrome (CS) in control and obese outpatients based on circadian rhythm and the overnight 1 mg dexamethasone (DEX) suppression test. Salivary cortisol was suppressed to less than 100 ng/dl after 1 mg DEX in control and obese patients. A single salivary cortisol measurement at 23:00 h and again after 1 mg DEX above the 90th percentile of the obese group values had sensitivity and specificity of 93 and 93% (23:00 h), and 91 and 94% (after DEX), respectively. The sensitivity improved to 100% when we combined both parameters. We also studied 11 CS children and 21 age-matched primary obese children for whom salivary cortisol sensitivity and specificity were 100/95% (23:00 h), and 100/95% (1 mg DEX), respectively. Similar to adults, sensitivity and specificity of 100% were obtained by combining 23:00 h and 1 mg DEX. The measurement of salivary cortisol is a useful tool for physiological studies and for the diagnosis of CS in children and adults on an outpatient basis.

Central Precocious Puberty that appears to be sporadic caused by Paternally inherited mutations in the imprinted GENE makorin ring finger 3

CONTEXT Loss-of-function mutations in makorin ring finger 3 (MKRN3), an imprinted gene located on the long arm of chromosome 15, have been recognized recently as a cause of familial central precocious puberty (CPP) in humans. MKRN3 has a potential inhibitory effect on GnRH secretion. OBJECTIVES The objective of the study was to investigate potential MKRN3 sequence variations as well as copy number and methylation abnormalities of the 15q11 locus in patients with apparently sporadic CPP. SETTING AND PARTICIPANTS We studied 215 unrelated children (207 girls and eight boys) from three university medical centers with a diagnosis of CPP. All but two of these patients (213 cases) reported no family history of premature sexual development. First-degree relatives of patients with identified MKRN3 variants were included for genetic analysis. MAIN OUTCOME MEASURES All 215 CPP patients were screened for MKRN3 mutations by automatic sequencing. Multiplex ligation-dependent probe amplification was performed in a partially overlapping cohort of 52 patients. RESULTS We identified five novel heterozygous mutations in MKRN3 in eight unrelated girls with CPP. Four were frame shift mutations predicted to encode truncated proteins and one was a missense mutation, which was suggested to be deleterious by in silico analysis. All patients with MKRN3 mutations had classical features of CPP with a median age of onset at 6 years. Copy number and methylation abnormalities at the 15q11 locus were not detected in the patients tested for these abnormalities. Segregation analysis was possible in five of the eight girls with MKRN3 mutations; in all cases, the mutation was inherited on the paternal allele. CONCLUSIONS We have identified novel inherited MKRN3 defects in children with apparently sporadic CPP, supporting a fundamental role of this peptide in the suppression of the reproductive axis.

Late-night Salivary Cortisol Has a Better Performance Than Urinary Free Cortisol in the Diagnosis of Cushing's Syndrome

CONTEXT The comparison of variability, reproducibility, and diagnostic performance of late-night salivary cortisol (LNSF) and urinary free cortisol (UFC) using concurrent and consecutive samples in Cushings syndrome (CS) is lacking. Objectives, Patients, and Methods: In a prospective study, we evaluated 3 simultaneous and consecutive samples of LNSF by RIA and UFC by liquid chromatography associated with tandem mass spectrometry in Cushings disease (CD) patients (n = 43), adrenal CS patients (n = 9), and obese subjects (n = 18) to compare their diagnostic performances. In CS patients, we also performed a modified CS severity index. RESULTS There was no difference in the coefficient of variation (percentage) between LNSF and UFC among the 3 samples obtained for each patient with Cushings disease (35 ± 26 vs 31 ± 24), adrenal CS (28 ± 14 vs 22 ± 14), and obesity (39 ± 37 vs 48 ± 20). LNSF confirmed the diagnosis of hypercortisolism even in the presence of normal UFC in 17.3% of CS, whereas the inverse situation was not observed for UFC. The area under the receiver-operating characteristic curves for LNSF was 0.999 (95% credible interval [CI] 0.990-1.00) and for UFC was 0.928 (95% CI 0.809-0.987). The ratio between areas under the curve was 0.928 (95% CI 0.810-0.988), indicating better performance of LNSF than UFC in diagnosing CS. There was no association between the CS severity index and the degree of biochemical hypercortisolism. CONCLUSION Our data show that despite similar variability between both methods, LNSF has a superior diagnostic performance than UFC and should be used as the primary biochemical diagnostic test for CS diagnosis.

A dose–response study of salivary cortisol after dexamethasone suppression test in Cushing's disease and its potential use in the differential diagnosis of Cushing's syndrome

objective  A dose–response study with different doses of dexamethasone (dex) to assess the corticotrophic resistance in Cushings disease (CD) using salivary cortisol as an end point has not yet been evaluated. We also reported our experience with salivary cortisol compared to plasma cortisol determination during dex suppression test (DST) and after ovine corticotrophin release hormone (oCRH) test in the differential diagnosis of Cushings syndrome (CS).