Real-time PCR quantitation of glucocorticoid receptor alpha isoform

BACKGROUND: The expression of glucocorticoid-receptor (GR) seems to be a key mechanism in the regulation of glucocorticoid (GC) sensitivity and is potentially involved in cases of GC resistance or hypersensitivity. The aim of this study is to describe a method for quantitation of GR alpha isoform (GRα) expression using real-time PCR (qrt-PCR) with analytical capabilities to monitor patients, offering standard-curve reproducibility as well as intra- and inter-assay precision. RESULTS: Standard-curves were constructed by employing standardized Jurkat cell culture procedures, both for GRα and BCR (breakpoint cluster region), as a normalizing gene. We evaluated standard-curves using five different sets of cell culture passages, RNA extraction, reverse transcription, and qrt-PCR quantification. Intra-assay precision was evaluated using 12 replicates of each gene, for 2 patients, in a single experiment. Inter-assay precision was evaluated on 8 experiments, using duplicate tests of each gene for two patients. Standard-curves were reproducible, with CV (coefficient of variation) of less than 11%, and Pearson correlation coefficients above 0,990 for most comparisons. Intra-assay and inter-assay were 2% and 7%, respectively. CONCLUSION: This is the first method for quantitation of GRα expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way

Impact of Early Postnatal Androgen Exposure on Voice Development

Background: the impact of early postnatal androgen exposure on female laryngeal tissue may depend on certain characteristics of this exposure. We assessed the impact of the dose, duration, and timing of early androgen exposure on the vocal development of female subjects who had been treated for adrenocortical tumor (ACT) in childhood.Methods: the long-term effects of androgen exposure on the fundamental vocal frequency (F0), vocal pitch, and final height and the presence of virilizing signs were examined in 9 adult (age, 18.4 to 33.5 years) and 10 adolescent (13.6 to 17.8 years) female ACT patients. We also compared the current values with values obtained 0.9 years to 7.4 years after these subjects had undergone ACT surgery, a period during which they had shown normal androgen levels.Results: of the 19 subjects, 17 (89%) had been diagnosed with ACT before 4 years of age, 1 (5%) at 8.16 years, and 1 (5%) at 10.75 years. Androgen exposure (2 to 30 months) was sufficiently strong to cause pubic hair growth in all subjects and clitoromegaly in 74% (14/19) of the subjects, but did not reduce their height from the target value. Although androgen exposure induced a remarkable reduction in F0 (132 Hz) and moderate pitch virilization in 1 subject and partial F0 virilization, resulting in F0 of 165 and 169 Hz, in 2 subjects, the majority had normal F0 ranging from 189 to 245 Hz.Conclusions: Female laryngeal tissue is less sensitive to androgen exposure between birth and adrenarche than during other periods. Differential larynx sensitivity to androgen exposure in childhood and F0 irreversibility in adulthood are age-, concentration-, duration-, and timing-dependent events that may also be affected by exposure to inhibitory or stimulatory hormones. Further studies are required to better characterize each of these factors.Brazilian State of Parana Secretary of Science, Technology and Higher Education (SETI)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Raul Carneiro Hospital Association for Childhood Protection (AHPIRC)American Lebanese Syrian Associated Charities (ALSAC)SETIAHPIRCALSACInst Pesquisa Pele Pequeno Principe, Curitiba, Parana, BrazilHosp Pequeno Principe, Curitiba, Parana, BrazilFac Pequeno Principe, Curitiba, Parana, BrazilInst Voz Maringa, Maringa, Parana, BrazilUniversidade Federal de São Paulo, Dept Fonoaudiol, São Paulo, BrazilClin Voz, Curitiba, Parana, BrazilIrmandade Santa Casa Misericordia São Paulo, São Paulo, BrazilUniv Fed Parana, Dept Saude Comunitaria, BR-80060000 Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Fonoaudiol, São Paulo, BrazilWeb of Scienc

AGK-BRAF gene fusion is a recurrent event in sporadic pediatric thyroid carcinoma

Thyroid cancer is the fastest increasing cancer worldwide in all age groups. Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer in both adults and children. PTC genomic landscape has been extensively studied in adults, but information regarding sporadic pediatric patients is lacking. Although BRAF V600E mutation is highly prevalent in adults, this mutation is uncommon in pediatric cases. As adult and pediatric PTC is a mitogen-activated protein kinase-driven cancer, this altered pathway might be activated by different genetic events. The aim of this study was to investigate the occurrence of AGK-BRAF fusion gene, recently described in radiation-exposed pediatric PTC, in a cohort of exclusively sporadic pediatric PTC. The series consisted of 30 pediatric PTC younger than 18 years of age at the time of diagnosis and 15 matched lymph node metastases (LNM). Primary tumors and matched LNM were screened for the presence of the AGK-BRAF fusion transcript by RT-PCR. To confirm the identity of the amplified products, randomly selected samples positive for the presence of the fusion transcripts were sequenced. Moreover, BRAF dual-color, break-apart probes confirmed BRAF rearrangement. Overall, the AGK-BRAF fusion gene was detected in 10% (3/30) of primary tumors. For one of these cases, paired LNM was also available, which also shows the presence of AGK-BRAF fusion gene. This study described, for the first time, the presence of AGK-BRAF in sporadic pediatric PTC. Understanding the molecular events underlying pediatric PTC may improve preoperative diagnosis, allow molecular prognostication and define a therapeutic approach toward sporadic PTC patients.Sao Paulo State Research Foundation (FAPESP)CNPqFAPESP scholarUniv Fed Sao Paulo, Dept Morphol & Genet, Div Genet, Genet Bases Thyroid Tumors Lab, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Pathol, Sao Paulo, SP, BrazilIrmandade Santa Casa Misericordia Sao Paulo, Dept Pediat, Sao Paulo, SP, BrazilIrmandade Santa Casa Misericordia Sao Paulo, Dept Med, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Morphol & Genet, Div Genet, Genet Bases Thyroid Tumors Lab, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Pathol, Sao Paulo, SP, BrazilFAPESP: 2012/02902-9FAPESP: 2013/03867-5FAPESP: 2014/06570-6CNPq: 470441/2013-5Web of Scienc

Insulin-like growth factor 1 gene (CA)n repeats and a variable number of tandem repeats of the insulin gene in Brazilian children born small for gestational age

OBJECTIVE: To investigate the influence of (CA)n repeats in the insulin-like growth factor 1 gene and a variable number of tandem repeats of the insulin gene on birth size in children who are small or adequate-sized for gestational age and to correlate these polymorphisms with serum insulin-like growth factor 1 levels and insulin sensitivity in children who are small for gestational age, with and without catch-up growth. PATIENTS AND METHODS: We evaluated 439 infants: 297 that were adequate-sized for gestational age and 142 that were small for gestational age (66 with and 76 without catch-up). The number of (CA)n repeat in the insulin-like growth factor 1 gene and a variable number of tandem repeats in the insulin gene were analyzed using GENESCAN software and polymerase chain reaction followed by enzymatic digestion, respectively. Clinical and laboratory data were obtained from all patients. RESULTS: The height, body mass index, paternal height, target height and insulin-like growth factor 1 serum levels were higher in children who were small for gestational age with catch-up. There was no difference in the allelic and genotypic distributions of both polymorphisms between the adequate-sized and small infants or among small infants with and without catch-up. Similarly, the polymorphisms were not associated with clinical or laboratory variables. CONCLUSION: Polymorphisms of the (CA)n repeats of the insulin-like growth factor 1 gene and a variable number of tandem repeats of the insulin gene, separately or in combination, did not influence pre- or postnatal growth, insulin-like growth factor 1 serum levels or insulin resistance

Reported shoes size during GH therapy: is foot overgrowth a myth or reality?

Objectives To describe population reference values for shoes size, and to identify possible disproportional foot growth during GH therapy.Materials and methods Construction of percentile chart based on 3,651 controls (male: 1,838; female: 1,813). The GH treated group included 13 children with idiopathic short stature (ISS) and 50 children with normal height, but with height prediction below their target height; male: 26 and female: 37 mean ± SD age 13.3 ± 1.9 and 12.9 ± 1.5 years, respectively. GH (0.05 mg/kg/day) was used for 3.2 ± 1.6 years, ranging from 1.0-10.3 years. Height expressed as SDS, target height (TH) SDS, self-reported shoes size and target shoes size (TSS) SDS were recorded.Results Reference values were established showed as a foot SDS calculator available online at www.clinicalcaselearning.com/v2. Definitive shoes size was attained in controls at mean age of 13y in girls and 14y in boys (average values 37 and 40, respectively). In the study group, shoes size was -0.15 ± 0.9 and -0.02 ± 1.3 SDS, with target feet of 0.08 ± 0.8 and -0.27 ± 0.7 SDS in males and females, respectively. There was a significant positive correlation between shoes size and familial TSS, between shoes size and height and between TSS and TH. There was no correlation between duration of GH treatment and shoes size. Our data suggest that during long-term treatment with GH, patients maintain proportional growth in shoes size and height, and the expected correlation with the familial target.Conclusions We conclude that there is no excessive increase in the size of foot as estimated by the size of shoes in individuals under long term GH therapy

Adrenal steroids synthesis during acute infectious diseases in infants

An increase in plasma 17OHP found in infants requiring differential diagnosis between septic shock and adrenal failure led us to look for adrenal steroids pattern during infection. Infants and Methods: 56 infants, 1-6 months old, were studied during infection of different degrees of severity. Plasma cortisol, 17OHP, androstenedione, DHEA, DHEA-S and testosterone were measured.Results: 24 patients showed an expected cortisol elevation. One child had a low cortisol level. The concentration of 17OHP was above 6.0 nmol/l (200 ng/dl) in 41 patients and above 30.2 nmol/l (1,000 ng/dl) in 10. Higher 17OHP levels and more severe diseases correlated positively.Conclusions: During infectious diseases some patients demonstrated not only cortisol elevation but also 17OHP as high as that observed in NC-CAH. We suggest that if 17OHP elevation is not characteristic of SL-CAH, glucocorticoid therapy should be started and an ACTH test should be performed after recovery before ruling out this pathology.Santa Casa Sao Paulo, Sch Med, Pediat Endocrinol Unit, Dept Pediat, Sao Paulo, BrazilHosp Municipal Infantil Menino Jesus, Pediat Intens Care Unit, Sao Paulo, BrazilUniv Sao Paulo, Sch Med, Dept Endocrinol, Endocrinol Unit Dev & Hormones, Sao Paulo, BrazilUniv Sao Paulo, Sch Med, Dept Endocrinol, Mol Genet Lab, Sao Paulo, BrazilWeb of Scienc