Epigenetic control of adamantinomatous craniopharyngiomas.

INTRODUCTION: Studies addressing the methylation pattern in adamantinomatous craniopharyngioma (ACP) are lacking. OBJECTIVE: To identify methylation signatures in ACPs regarding clinical presentation and outcome. METHODS: Clinical and pathology data were collected from 35 ACP patients (54% male; 18.1 years [2-68]). CTNNB1 mutations and methylation profile (MethylationEPIC/Array-Illumina) were analyzed in tumoral DNA. Unsupervised machine learning analysis of this comprehensive methylome sample was achieved using hierarchical clustering and multi-dimensional scaling. Statistical associations between clusters and clinical features were achieved using Fisher's test and global biological process interpretations were aided by Gene Ontology enrichment analyses. RESULTS: Two clusters were revealed consistently by all unsupervised methods (ACP-1: n = 18; ACP-2: n = 17) with strong bootstrap statistical support. ACP-2 was enriched by CTNNB1 mutations (100% vs 56%, P = 0.0006), hypomethylated in CpG Island (CGI), non-CGI sites, and globally (P < 0.001), and associated with greater tumor size (24.1 vs 9.5cm3, P = 0.04). Enrichment analysis highlighted pathways on signaling transduction, transmembrane receptor, development of anatomical structures, cell-adhesion, cytoskeleton organization, and cytokine binding, and also cell-type specific biological processes as regulation of oligodendrocytes, keratinocyte, and epithelial cells differentiation. CONCLUSION: Two clusters of ACP patients were consistently revealed by unsupervised machine learning methods, being one of them significantly hypomethylated, enriched by CTNNB1 mutated ACPs, and associated with increased tumor size. Enrichment analysis reinforced pathways involved in tumor proliferation and in cell-specific tumoral microenvironment.

Nicotinamide Nucleotide Transhydrogenase Is Essential for Adrenal Steroidogenesis: Clinical and In Vitro Lessons.

CONTEXT: Nicotinamide nucleotide transhydrogenase (NNT) acts as an antioxidant defense mechanism. NNT mutations cause familial glucocorticoid deficiency (FGD). How impaired oxidative stress disrupts adrenal steroidogenesis remains poorly understood. OBJECTIVE: To ascertain the role played by NNT in adrenal steroidogenesis. METHODS: The genotype-phenotype association of a novel pathogenic NNT variant was evaluated in a boy with FGD. Under basal and oxidative stress (OS) induced conditions, transient cell cultures of the patient's and controls' wild-type (WT) mononuclear blood cells were used to evaluate antioxidant mechanisms and mitochondrial parameters (reactive oxygen species [ROS] production, reduced glutathione [GSH], and mitochondrial mass). Using CRISPR/Cas9, a stable NNT gene knockdown model was built in H295R adrenocortical carcinoma cells to determine the role played by NNT in mitochondrial parameters and steroidogenesis. NNT immunohistochemistry was assessed in fetal and postnatal human adrenals. RESULTS: The homozygous NNT p.G866D variant segregated with the FGD phenotype. Under basal and OS conditions, p.G866D homozygous mononuclear blood cells exhibited increased ROS production, and decreased GSH levels and mitochondrial mass than WT NNT cells. In line H295R, NNT knocked down cells presented impaired NNT protein expression, increased ROS production, decreased the mitochondrial mass, as well as the size and the density of cholesterol lipid droplets. NNT knockdown affected steroidogenic enzyme expression, impairing cortisol and aldosterone secretion. In human adrenals, NNT is abundantly expressed in the transition fetal zone and in zona fasciculata. CONCLUSION: Together, these studies demonstrate the essential role of NNT in adrenal redox homeostasis and steroidogenesis.

Vitamin D receptor activation is a feasible therapeutic target to impair adrenocortical tumorigenesis.

OBJECTIVE: To evaluate the therapeutic potential of vitamin D receptor (VDR) signaling in adrenocortical carcinoma (ACC) cells. METHODS: We evaluated VDR's methylation pattern in H295R ACC cells, and investigated the effects of calcitriol and seocalcitol treatments on adrenocortical tumorigenesis. RESULTS: VDR was hypermethylated and underexpressed in basal H295R cells. Treatments with calcitriol and seocalcitol restored VDR signaling, resulted in antiproliferative effects, and impaired Wnt/B-catenin signaling. RNAseq of treated cells demonstrated VDR activation on steroid hormones biosynthesis and Rap1 signaling, among others. In vivo, seocalcitol constrained the growth of H295R xenografts and reduced autonomous tumor steroid secretion without hypercalcemia-associated side effects. CONCLUSIONS: H295R cells present VDR hypermethylation, which can be responsible for its underexpression and signaling inactivation under basal conditions. VDR signaling promoted antiproliferative effects in vitro and in vivo, suggesting that it may be a potential therapeutic target for ACC and a valuable tool for patient's clinical management.

MicroRNA-149-3p expression correlates with outcomes of adrenocortical tumor patients and affects proliferation and cell cycle progression of H295A adrenocortical cancer cell line.

Pediatric adrenocortical tumor (ACT) is a rare and aggressive neoplasm, with incidence in southern and southeastern Brazil 10-15 times higher than worldwide. Although microRNAs (miRNAs) have been reported to act as tumor suppressors or oncogenes in several cancers, the role of miR-149-3p in ACT remains unknown. In this study, we evaluated the expression of miR-149-3p in 67 pediatric ACT samples and 19 non-neoplastic adrenal tissues. The overexpression of miR-149-3p was induced in H295A cell line, and cell viability, proliferation, colony formation, and cell cycle were assessed by in miR-149-3p mimic or mimic control. In silico analysis were used to predict miR-149-3p putative target genes. CDKN1A expression at the mRNA and protein levels was evaluated by qRT-PCR and western blot, respectively. Higher miR-149-3p expression was associated with unfavorable ACT outcomes. Compared to the mimic control, miR-149-3p overexpression increased cell viability and colony formation, and affected cell cycle progression. Also, we identified CDKN1A as a potential miR-149-3p target gene, with decreased expression at both the gene and protein levels in miR-149-3p mimic cells. Collectively, these findings suggest that miR-149-3p promotes H295A cell viability by downregulating CDKN1A and provide evidence that miR-149-3p may be useful as a novel therapeutic target for pediatric ACT.

Telomere length and Wnt/ß-catenin pathway in adamantinomatous craniopharyngiomas.

Objectives: To evaluate how telomere length behaves in adamantinomtous craniopharyngioma (aCP) and if it contributes to the pathogenesis of aCPs with and without CTNNB1 mutations. Design: Retrospective cross-sectional study enrolling 42 aCP patients from 2 tertiary institutions. Methods: 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. Results: 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. Conclusions: CTNNB1: 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/ß-catenin pathway and telomere biology in the pathogenesis of aCPs.

Vitamin D receptor hypermethylation as a biomarker for pediatric adrenocortical tumors.

Objective: 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. Design: Retrospective cross-sectional study enrolling pediatric patients with ACT from two tertiary referral institutions. Methods: 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. Results: 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. Conclusions: 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.

MicroRNA expression profile predicts prognosis of pediatric adrenocortical tumors.

Pediatric adrenocortical tumors (ACT) are rare aggressive neoplasms with heterogeneous prognosis. Despite extensive efforts, identifying reliable prognostic factors for pediatric patients with ACT remains a challenge. MicroRNA (miRNA) signatures have been associated with cancer diagnosis, treatment response, and prognosis of several types of cancer. However, the role of miRNAs has been poorly explored in pediatric ACT. In this study, we performed miRNA microarray profiling on a cohort of 37 pediatric ACT and nine nonneoplastic adrenal (NNA) samples and evaluated the prognostic significance of abnormally expressed miRNAs using Kaplan-Meier plots, log-rank test, and Cox regression analysis. We identified a total of 98 abnormally expressed miRNAs; their expression profile discriminated ACT from NNAs. Among the 98 deregulated miRNAs, 17 presented significant associations with patients' survival. In addition, higher expression levels of hsa-miR-630, -139-3p, -125a-3p, -574-5p, -596, -564, -1321, and -423-5p and lower expression levels of hsa-miR-377-3p, -126-3p, -410, -136-3p, -29b-3p, -29a-3p, -337-5p, -143-3p, and 140-5p were significantly associated with poor prognosis, tumor relapse, and/or death. Importantly, the expression profile of these 17 miRNAs stratified patients into two groups of ACTs with different clinical outcomes. Although some individual miRNAs exhibit potential prognostic values in ACTs, only the 17 miRNA-based expression clustering was considered an independent prognostic factor for 5-year event-free survival (EFS) compared to other clinicopathological features. In conclusion, our study reports for the first time associations between miRNA profiles and childhood ACT prognosis, providing evidence that miRNAs could be useful biomarkers to discriminate patients with favorable and unfavorable clinical outcomes.

TCF21/POD-1, a Transcritional Regulator of SF-1/NR5A1, as a Potential Prognosis Marker in Adult and Pediatric Adrenocortical Tumors.

With recent progress in understanding the pathogenesis of adrenocortical tumors (ACTs), identification of molecular markers to predict their prognosis has become possible. Transcription factor 21 (TCF21)/podocyte-expressed 1 (POD1) is a transcriptional regulatory protein expressed in mesenchymal cells at sites of epithelial-mesenchymal transition during the development of different systems. Adult carcinomas express less TCF21 than adenomas, in addition, the KEGG pathway analysis has shown that BUB1B, among others genes, is negatively correlated with TCF21 expression. The difference between BUB1B and PTEN-induced putative kinase 1 (PINK1) expression has been described previously to be associated with survival in adult but not in pediatric carcinomas. Here, we analyzed the gene expression of TCF21, BUB1B, PINK1, and NR5A1 in adult and pediatric ACTs. We found a negative correlation between the relative expression levels of TCF21 and BUB1B in adult ACTs, but the relative expression levels of TCF21, BUB1B, PINK1, and NR5A1 were similar in childhood ACTs. In addition, we propose using the subtracted expression levels of the TCF21/POD-1 genes as a predictor of overall survival (OS) in adult carcinomas and TCF21-NR5A1 as a predictor of malignancy for pediatric tumors in patients aged <5 years. These results require further validation in different cohorts of both adult and pediatric samples. Finally, we observed that the OS for patients aged <5 years was markedly favorable compared with that for patients >5 years as well as adult patients with carcinoma. In summary, we propose TCF21/POD-1 as a new prognostic marker in adult and pediatric ACTs.

Homocysteine, folate, hs-C-reactive protein, tumor necrosis factor alpha and inflammatory proteins: are these biomarkers related to nutritional status and cardiovascular risk in childhood-onset systemic lupus erythematosus?

BACKGROUND: Childhood-onset systemic lupus erythematosus (c-SLE) is a chronic autoimmune disease which increases cardiovascular risk factors (CRF) such as elevated homocysteine, TNF-α, and hs-C reactive protein. METHODS: We evaluated BMI, waist circumference (WC), 24-h recalls, SLEDAI-2 K, SLICC/ACR-DI, serum levels of homocysteine, folate, TNF-α, hs-C reactive protein, lipid profile, proteomic data, and duration of corticosteroid therapy in 19 c-SLE and 38 healthy volunteers. Physiological and anthropometric variables of c-SLE and healthy controls were compared by ANCOVA. k-cluster was used to separate c-SLE into two different groups with the best and the worst metabolic profile according to previous analysis showing some metabolites that were statistically different from controls, such as homocysteine, TNF-α, hs-CRP and folate levels. These two clusters were again compared with the control group regarding nutritional parameters, lipid profile and also proteomic data. RESULTS: Individuals with c-SLE presented higher BMI, WC, homocysteine, triglycerides, TNF-α, hs-CRP and lower folate levels when compared to controls. We found 10 proteins whose relative abundances were statistically different between control group and lupus clusters with the best (LCBMP) and the worst metabolic profile (LCWMP). A significant positive correlation was found between TNF-α and triglycerides and between hs-CRP and duration of corticosteroid therapy. CONCLUSION: Cardiovascular disease (CVD) risk parameters were worse in c-SLE. A less protective CVD proteomic profile was found in LCWMP.

TCF21/POD-1, a transcritional regulator of SF-1/NR5A1, as a potential prognosis marker in adult and pediatric adrenocortical tumors

With recent progress in understanding the pathogenesis of adrenocortical tumors (ACTs), identification of molecular markers to predict their prognosis has become possible. Transcription factor 21 (TCF21)/podocyte-expressed 1 (POD1) is a transcriptional regulatory protein expressed in mesenchymal cells at sites of epithelial-mesenchymal transition during the development of different systems. Adult carcinomas express less TCF21 than adenomas, in addition, the KEGG pathway analysis has shown that BUB1B, among others genes, is negatively correlated with TCF21 expression. The difference between BUB1B and PTEN-induced putative kinase 1 (PINK1) expression has been described previously to be associated with survival in adult but not in pediatric carcinomas. Here, we analyzed the gene expression of TCF21, BUB1B, PINK1, and NR5A1 in adult and pediatric ACTs. We found a negative correlation between the relative expression levels of TCF21 and BUB1B in adult ACTs, but the relative expression levels of TCF21, BUB1B, PINK1, and NR5A1 were similar in childhood ACTs. In addition, we propose using the subtracted expression levels of the TCF21/POD-1 genes as a predictor of overall survival (OS) in adult carcinomas and TCF21-NR5A1 as a predictor of malignancy for pediatric tumors in patients aged <5 years. These results require further validation in different cohorts of both adult and pediatric samples. Finally, we observed that the OS for patients aged <5 years was markedly favorable compared with that for patients >5 years as well as adult patients with carcinoma. In summary, we propose TCF21/POD-1 as a new prognostic marker in adult and pediatric ACTs.

TCF21/POD-1, a transcritional regulator of SF-1/NR5A1, as a potential prognosis marker in adult and pediatric adrenocortical tumors

With recent progress in understanding the pathogenesis of adrenocortical tumors (ACTs), identification of molecular markers to predict their prognosis has become possible. Transcription factor 21 (TCF21)/podocyte-expressed 1 (POD1) is a transcriptional regulatory protein expressed in mesenchymal cells at sites of epithelial-mesenchymal transition during the development of different systems. Adult carcinomas express less TCF21 than adenomas, in addition, the KEGG pathway analysis has shown that BUB1B, among others genes, is negatively correlated with TCF21 expression. The difference between BUB1B and PTEN-induced putative kinase 1 (PINK1) expression has been described previously to be associated with survival in adult but not in pediatric carcinomas. Here, we analyzed the gene expression of TCF21, BUB1B, PINK1, and NR5A1 in adult and pediatric ACTs. We found a negative correlation between the relative expression levels of TCF21 and BUB1B in adult ACTs, but the relative expression levels of TCF21, BUB1B, PINK1, and NR5A1 were similar in childhood ACTs. In addition, we propose using the subtracted expression levels of the TCF21/POD-1 genes as a predictor of overall survival (OS) in adult carcinomas and TCF21-NR5A1 as a predictor of malignancy for pediatric tumors in patients aged <5 years. These results require further validation in different cohorts of both adult and pediatric samples. Finally, we observed that the OS for patients aged <5 years was markedly favorable compared with that for patients >5 years as well as adult patients with carcinoma. In summary, we propose TCF21/POD-1 as a new prognostic marker in adult and pediatric ACTs.

IGF2 and IGF1R in pediatric adrenocortical tumors: roles in metastasis and steroidogenesis.

Deregulation of the IGF system observed in human tumors indicates a role in malignant cell transformation and in tumor cell proliferation. Although overexpression of the IGF2 and IGF1R genes was described in adrenocortical tumors (ACTs), few studies reported their profiles in pediatric ACTs. In this study, the IGF2 and IGF1R expression was evaluated by RT-qPCR according to the patient's clinical/pathological features in 60 pediatric ACT samples, and IGF1R protein was investigated in 45 samples by immunohistochemistry (IHC). Whole transcriptome and functional assays were conducted after IGF1R inhibition with OSI-906 in NCI-H295A cell line. Significant IGF2 overexpression was found in tumor samples when compared with non-neoplastic samples (P<0.001), significantly higher levels of IGF1R in patients with relapse/metastasis (P=0.031) and moderate/strong IGF1R immunostaining in 62.2% of ACTs, but no other relationship with patient survival and clinical/pathological features was observed. OSI-906 treatment downregulated genes associated with MAPK activity, induced limited reduction of cell viability and increased the apoptosis rate. After 24h, the treatment also decreased the expression of genes related to the steroid biosynthetic process, the protein levels of the steroidogenic acute regulatory protein (STAR), and androgen secretion in cell medium, supporting the role of IGF1R in steroidogenesis of adrenocortical carcinoma cells. Our data showed that the IGF1R overexpression could be indicative of aggressive ACTs in children. However, in vitro treatments with high concentrations of OSI-906 (>1µM) showed limited reduction of cell viability, suggesting that OSI-906 alone could not be a suitable therapy to abolish carcinoma cell growth.

[Human milk for neonatal pain relief during ophthalmoscopy]. / O leite humano no alívio da dor neonatal no exame de fundo de olho.

Ophthalmoscopy performed for the early diagnosis of retinopathy of prematurity (ROP) is painful for preterm infants, thus necessitating interventions for minimizing pain. The present study aimed to establish the effectiveness of human milk, compared with sucrose, for pain relief in premature infants subjected to ophthalmoscopy for the early diagnosis of ROP. This investigation was a pilot, quasi-experimental study conducted with 14 premature infants admitted to the neonatal intensive care unit (NICU) of a university hospital. Comparison between the groups did not yield a statistically significant difference relative to the crying time, salivary cortisol, or heart rate (HR). Human milk appears to be as effective as sucrose in relieving acute pain associated with ophthalmoscopy. The study's limitations included its small sample size and lack of randomization. Experimental investigations with greater sample power should be performed to reinforce the evidence found in the present study.

Human milk for neonatal pain relief during ophthalmoscopy / La leche humana en el alivio del dolor neonatal en el examen de fondo de ojo / O leite humano no alívio da dor neonatal no exame de fundo de olho

Ophthalmoscopy performed for the early diagnosis of retinopathy of prematurity (ROP) is painful for preterm infants, thus necessitating interventions for minimizing pain. The present study aimed to establish the effectiveness of human milk, compared with sucrose, for pain relief in premature infants subjected to ophthalmoscopy for the early diagnosis of ROP. This investigation was a pilot, quasi-experimental study conducted with 14 premature infants admitted to the neonatal intensive care unit (NICU) of a university hospital. Comparison between the groups did not yield a statistically significant difference relative to the crying time, salivary cortisol, or heart rate (HR). Human milk appears to be as effective as sucrose in relieving acute pain associated with ophthalmoscopy. The study’s limitations included its small sample size and lack of randomization. Experimental investigations with greater sample power should be performed to reinforce the evidence found in the present study. .

El examen de fondo de ojo para el diagnóstico precoz de retinopatía prematura causa dolor en el prematuro, siendo necesarias intervenciones que la minimicen. El objetivo fue investigar la efectividad de la leche humana para el alivio del dolor en los prematuros sometidos al examen de fondo de ojo para el diagnóstico precoz de la enfermedad, en comparación con la sacarosa. Estudio piloto de carácter cuasi-experimental, realizado con 14 prematuros internados en la unidad neonatal de un hospital universitario. En la comparación entre los grupos, no hubo diferencia estadística significativa relacionada a la duración del tiempo del llanto, la concentración del cortisol salival y la frecuencia cardiaca. La leche humana parece ser tan efectiva como la sacarosa en el alivio del dolor agudo debido al examen. Una limitación fue el reducido tamaño de la muestra y la falta de aleatorización. Es necesaria la realización de estudios experimentales con mayor tamaño de muestra para fortalecer las evidencias encontradas.

O exame de fundo de olho para o diagnóstico precoce da retinopatia da prematuridade causa dor no prematuro, sendo necessárias intervenções que a minimizem. O objetivo foi investigar a efetividade do leite humano para alívio da dor em prematuros submetidos ao exame de fundo de olho para diagnóstico precoce da doença, em comparação com a sacarose. Tratou-se de um estudo piloto de caráter quase experimental, realizado com 14 prematuros internados na unidade neonatal de um hospital universitário. Na comparação entre os grupos, não houve diferença estatística significativa relacionada à duração do tempo de choro, concentração do cortisol salivar e frequência cardíaca. O leite humano parece ser tão efetivo quanto a sacarose no alívio da dor aguda relacionada ao exame. Tem-se como limitação o tamanho reduzido da amostra e a falta de randomização. É necessária a realização de estudos experimentais com maior poder amostral para fortalecer as evidências encontradas.

Spindle assembly checkpoint gene expression in childhood adrenocortical tumors (ACT): Overexpression of Aurora kinases A and B is associated with a poor prognosis.

BACKGROUND: Pediatric adrenocortical tumors (ACT) are rare malignancies and treatment has a small impact on survival in advanced disease and the discovery of potential target genes could be important in new therapeutic approaches. METHODS: The mRNA expression levels of spindle checkpoint genes AURKA, AURKB, BUB, and BUBR1 were analyzed in 60 children with ACT by quantitative real time PCR. The anticancer effect of ZM447439, an experimental AURK inhibitor, was analyzed in a primary childhood ACT culture carrying the TP53 p.R337H mutation. RESULTS: A significant association was observed between malignancy as defined by Weiss score ≥3 and higher AURKA (2.0-fold, P = 0.01), AURKB (7.0-fold, P = 0.007), and BUBR1 (5.8-fold, P = 0.007) gene expression, and between unfavorable event (death or relapse) and higher expression of AURKA (6.0-fold, P = 0.034) and AURKB (17-fold, P = 0.013). Overexpression of AURKA and AURKB was associated with lower event-free survival in uni- (P < 0.001 and P = 0.006, respectively) and multivariate (P = 0.002 and P = 0.03, respectively) analysis. Significant lower Event free survival (EFS) was also observed in patients with moderate/strong immunostaining to AURKA (P = 0.012) and AURKB (P = 0.045). ZM447439 was able to induce inhibition of proliferation and colony formation in a primary childhood ACT culture carrying the TP53 p.R337H mutation. CONCLUSION: Our results suggest that AURKA and AURKB overexpression in pediatric ACT may be related to more aggressive disease and the inhibition of these proteins could be an interesting approach for the treatment of these tumors.

Measurement of salivary cortisol as a marker of stress in newborns in a neonatal intensive care unit.

AIMS: The study was designed to evaluate the newborn (NB) stress response during the inpatient time in the neonatal intensive care unit. METHODS: A quantitative, prospective, observational study was conducted with two NB groups. The first group consisted of 12 NB patients in the neonatal intensive care unit as the experimental group (EG), and the second included 43 NBs who were sent to their own homes and were considered the control group (CG). The EG's salivary cortisol concentration was measured on the 2nd day (D2) and 9th day (D9) of life. The CG's salivary cortisol concentration was measured on the 14th day of life at the child's own home. RESULTS: The salivary cortisol concentration levels for the EG on D2 and D9 and for the CG were 4.3151 ± 2.6492, 1.826 ± 1.2252, and 1.0166 ± 0.8300 ng/dl, respectively. These findings indicated the presence of an adrenal response to stress during the first inpatient days. CONCLUSIONS: The salivary cortisol concentration is an accurate method to indicate neonatal stress. The glucocorticoids frequently used in the prenatal period suppress the adrenal glands and interfere with the stress response.

A rational, non-radioactive strategy for the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

CONTEXT: Molecular diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) has not been straightforward. OBJECTIVE: To conduct a comprehensive genetic analysis by Multiplex Ligation dependent Probe Amplification (MLPA) and evaluate its reliability for the molecular CAH-21OHD diagnosis. PATIENTS AND METHODS: We studied 99 patients from 90 families with salt-wasting (SW; n=32), simple-virilizing (SV; n=29), and non-classical (NC; n=29) CAH-21OHD. Molecular analysis was sequentially performed by detecting the most frequent point mutations by allele-specific oligonucleotide polymerase chain reaction (ASO-PCR), large rearrangements by MLPA, and rare mutations by direct sequencing. Parental segregation was evaluated. RESULTS: ASO-PCR detected microconversions in 164 alleles (91.1%). MLPA identified CYP21A1P large conversions to CYP21A2 in 7 of the remaining 16 (43.7%), 30-kb deletions including the 3'-end of CYP21A1P, C4B, and the 5'-end of CYP21A2 in 3 of the 16 (18.7%), and a complete CYP21A2 deletion in one (6.3%). Five alleles (2.7%) required direct sequencing; three mutations located in the CYP21A2 gene and two derived from CYP21A1P were found. No parental segregation was observed in patients with the c.329_336del and/or the CL6 cluster mutations. These cases were not diagnosed by ASO-PCR, but MLPA detected deletions in the promoter region of the CYP21A2 gene, explaining the genotype/phenotype dissociation. CONCLUSION: Using the proposed algorithm, all alleles were elucidated. False-positive results in MLPA occurred when mutations or polymorphisms were located close to the probe-binding regions. These difficulties were overcome by the association of MLPA with ASO-PCR and paternal segregation. Using these approaches, we can successfully use MLPA in a cost-effective laboratory routine for the molecular diagnosis of CAH-21OHD.

Expression profile of apoptosis-related genes in childhood adrenocortical tumors: low level of expression of BCL2 and TNF genes suggests a poor prognosis.

BACKGROUND: Impaired apoptosis has been implicated in the development of childhood adrenocortical tumors (ACT), although the expression of apoptosis-related gene expression in such tumors has not been reported. METHODS: The mRNA expression levels of the genes CASP3, CASP8, CASP9, FAS, TNF, NFKB, and BCL2 were analyzed by quantitative real-time PCR in consecutive tumor samples obtained at diagnosis from 60 children with a diagnosis of ACT and in 11 non-neoplastic adrenal samples. BCL2 and TNF protein expression was analyzed by immunohistochemistry. RESULTS: A significant association was observed between tumor size ≥100 g and lower expression levels of the BCL2 (P=0.03) and TNF (P=0.05) genes; between stage IV and lower expression levels of CASP3 (P=0.008), CASP9 (P=0.02), BCL2 (P=0.002), TNF (P=0.05), and NFKB (P=0.03); Weiss score ≥3 and lower expression of TNF (P=0.01); unfavorable event and higher expression values of CASP9 (P=0.01) and lower values of TNF (P=0.02); and death and lower expression of BCL2 (P=0.04). Underexpression of TNF was associated with lower event-free survival in uni- and multivariate analyses (P<0.01). Similar results were observed when patients with Weiss score <3 were excluded. CONCLUSION: This study supports the participation of apoptosis-related genes in the biology and prognosis of childhood ACT and suggests the complex role of these genes in the pathogenesis of this tumor.