Canalopatias em endocrinologia: achados genéticos recentes e fisiopatologia

Ion channels serve diverse cellular functions, mainly in cell signal transduction. In endocrine cells, these channels play a major role in hormonal secretion, Ca2+-mediated cell signaling, transepithelial transport, cell motility and growth, volume regulation and cellular ionic content and acidification of lysosomal compartments. Ion channel dysfunction can cause endocrine disorders or endocrine-related manifestations, such as pseudohypoaldosteronism type 1, Liddle syndrome, Bartter syndrome, persistent hyperinsulinemic hypoglycemia of infancy, neonatal diabetes mellitus, cystic fibrosis, Dent's disease, hypomagnesemia with secondary hipocalcemia, nephrogenic diabetes insipidus and, the most recently genetically identified channelopathy, thyrotoxic hypokalemic periodic paralysis. This review briefly recapitulates the membrane action potential in endocrine cells and offers a short overview of known endocrine channelopathies with focus on recent progress regarding the pathophysiological mechanisms and functional genetic defects.Canais iônicos auxiliam diferentes funções celulares, principalmente na transdução de sinal. Nas células endócrinas, esses canais têm funções importantes na secreção hormonal, sinalização do Ca2+, transporte transepitelial, regulação da motilidade, volume e conteúdo iônico celular e da acidificação do compartimento lisossomal (pH). Como esperado, as alterações nos canais iônicos podem causar distúrbios endocrinológicos, como pseudo-hipoaldosteronismo tipo 1, síndrome de Liddle, síndrome de Bartter, hipoglicemia hiperinsulinêmica da infância, diabetes melito neonatal, fibrose cística, doença de Dent, hipomagnesemia com hipocalcemia secundária, diabetes insípido nefrogênico e paralisia periódica tirotóxica hipocalêmica. Este artigo propõe uma breve revisão das canalopatias endócrinas conhecidas, com foco particular nos recentes progressos no conhecimento dos mecanismos fisiopatológicos adquirido a partir das alterações funcionais encontradas.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MedicinaUNIFESP-EPM Departamento de BioquímicaUNIFESP, EPM, Depto. de MedicinaUNIFESP, EPM Depto. de BioquímicaSciEL

Androgens by immunoassay and mass spectrometry in children with 46,XY disorder of sex development

Objective: Steroid measurement is a challenge in pediatric endocrinology. Currently, liquid chromatography with tandem mass spectrometry (LC-MS/MS) is considered a gold standard for this purpose. The aim of this study was to compare both LC-MS/MS and immunoassay (IA) for androgens before and after human recombinant chorionic gonadotropin (rhCG) stimulus in children with 46,XY disorders of sex development (DSD). Methods: Nineteen patients with 46,XY DSD were evaluated; all of them were prepubertal and non-gonadectomized. Testosterone, dihydrotestosterone (DHT), DHEA and androstenedione were measured by IA and LC-MS/MS before and 7 days after rhCG injection. The correlation between IA and LC-MS/MS was analyzed by the intraclass correlation coefficient (ICC) and Spearman’s rank correlation coefficient (SCC). For concordance analysis the Passing and Bablok (PB) regression and the Bland and Altman (BA) method were used. Results: Testosterone showed excellent correlation (ICC = 0.960 and SCC = 0.964); DHT showed insignificant and moderate correlations as indicated by ICC (0.222) and SCC (0.631), respectively; DHEA showed moderate correlation (ICC = 0.585 and SCC = 0.716); and androstenedione had poor and moderate correlations in ICC (0.363) and SCC (0.735), respectively. Using the PB method, all hormones showed a linear correlation, but proportional and systematic concordance errors were detected for androstenedione, systematic errors for testosterone and no errors for DHEA and DHT. By the BA method, there was a trend of IA to overestimate testosterone and androstenedione and underestimate DHEA and DHT when compared to LC-MS/MS. Conclusion: Traditional IA should be replaced by LC-MS/MS for the androgens measurement in prepubertal children whenever is possible

The absence of mutations in homeobox candidate genes HOXA3, HOXB3, HOXD3 and PITX2 in familial and sporadic thyroid hemiagenesis

Background: the molecular mechanisms leading to the formation of the two thyroid symmetrical lobes, which are impaired in thyroid hemiagenesis (TH), are little known. Objective: the aim of this work was to search for mutations in thyroid developmental candidate genes HOXA3, HOXB3, HOXD3 and PITX2.Methods: Total DNA from peripheral blood was extracted and then the entire coding region of all these genes was amplified by polymerase chain reaction and direct sequencing.Results: Herein we describe familial cases of TH in two generations (proband and his father), in addition to other two sporadic cases. We have found polymorphisms in the HOXB3 (rs2229304), HOXD3 (rs34729309, rs1051929, c. 543199G > T and c. 543-34G > A; and a new synonymous variant, NP_ 008829.3: p. 314; C > G) and PITX2 (c. 45+ 76C > T) genes, but no deleterious mutations.Conclusion: These results suggest the existence of other left-right thyroid asymmetry candidate genes in humans such as classical Mendelian mutation-causing disease, as well as other etiopathogenic mechanisms such as epigenetic modifications, especially for sporadic hemiagenesis.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Lab Mol & Translat Endocrinol, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Lab Mol & Translat Endocrinol, Dept Med, Escola Paulista Med, BR-04039032 São Paulo, BrazilUniv Fed Parana, Dept Pediat, BR-80060000 Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Lab Mol & Translat Endocrinol, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Lab Mol & Translat Endocrinol, Dept Med, Escola Paulista Med, BR-04039032 São Paulo, BrazilFAPESP: 2012/01628-0FAPESP: 2012/02465-8FAPESP: 2011/20747-8FAPESP: 2012/00079-3Web of Scienc

Clinical and Molecular Analysis of Thyroid Hypoplasia: A Population-Based Approach in Southern Brazil

Background: Congenital hypothyroidism (CH) is mainly due to developmental abnormalities leading to thyroid dysgenesis (TD). TD encompasses very distinct morphologic subtypes of disease. This study examined and compared the phenotype in TD variants and searched for genetic alterations in sporadic thyroid hypoplasia (TH), the most misdiagnosed form of CH. This was a longitudinal study over a 14-year period (1990-2004).Methods: A continuous series of 353 children with TD was identified using thyroid function tests [thyroxine (T4) and TSH], scintigraphy, and ultrasound as diagnostic tools. Individual phenotypes were analyzed in 253 children with TD. Mutations in the most likely candidate genes were studied in 35 cases of TH.Results: the overall birth prevalence of permanent CH was 1:4795. Ectopy represented 37% of all cases of permanent primary CH, dyshormonogenesis 28%, agenesis 24%, hypoplasia 10%, and hemiagenesis 1%. the lowest screening T4 level and the highest TSH level were in the agenetic group, followed by TH. the TH group had an improvement in the thyroid function showing less-severe phenotype with aging. in the molecular analysis, one patient was identified with a mutation in the PAX8 gene (155G>C; R52P); four patients had a heterozygous G>C substitution in position -569; two patients showed a (234C>A; P52T) or (2181C>G; D727E) polymorphic variants of the TSH-R gene; and one patient presented a novel heterozygous nonsynonymous substitution, 293G>A; S98N, in the NKX2.5 gene.Conclusions: the prevalence of CH was within the previously reported range of 1:3000-4000. Ectopy was the most common etiology. Clinical analysis revealed distinct hormonal patterns in TH subgroup when compared with other variants of TD, with genetic abnormalities identified only in few cases in the TSH-R, PAX8, and NKX2.5 genes.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao Araucaria, grant Programa de PesquisaUniversidade Federal de São Paulo, Mol Endocrinol Lab, Dept Med, Div Endocrinol, BR-04039032 São Paulo, BrazilUniv Fed Parana, Div Endocrinol & Metab, BR-80060000 Curitiba, Parana, BrazilUniv Fed Parana, Pediat Endocrinol Unit, BR-80060000 Curitiba, Parana, BrazilNeonatal Screening Program Fundacao Ecumen Protec, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Mol Endocrinol Lab, Dept Med, Div Endocrinol, BR-04039032 São Paulo, BrazilFAPESP: 06/54950-6Web of Scienc

Novel immunoassay for TSH measurement in rats

ABSTRACT Measuring thyroid hormones is an important aspect for the study of metabolism and for monitoring diseases in both human and animal models. The traditional method for hormone measurement in rats is the radioimmunoassay (RIA). However, the RIA is associated with some practical disadvantages, including the use of radioactive material, the need for specialized equipment and expert staff, the short shelf-life of kits according to the half-life of the radioisotope and high costs. The objective of this study was to develop a new cost-effective method for measuring TSH levels in rats that avoids the use of radioactive material. We developed an in-house competitive immunoassay using a reference standard, polyclonal antibody produced in rabbits and biotinylated antigen. This method was tested in 64 Wistar rats that were divided into a control group (n = 41) and a group with hypothyroidism (n = 23). Our assay demonstrated an analytical sensitivity of 0.24 ng/mL (n = 12) and an intra-assay coefficient of variation (CV) of 8.9% for sera with TSH levels of 1.5 ng/mL and 13.2% for sera with TSH levels of 17.5 ng/mL (n = 14). The inter-assay CV was 13.5% for sera with TSH levels of 1.4 ng/mL and 14.5% for TSH levels of 18.2 ng/mL (n = 5). The analysis of mean TSH levels in control rats (5.06 ± 0.5701) and hypothyroid rats (51.09 ± 5.136) revealed a statistically significant difference (p < 0.001) between the groups. This method showed good sensitivity, can be automated and is low-cost compared with RIA. Our method offers a viable alternative for TSH measurement in rats

Androgens by immunoassay and mass spectrometry in children with 46,XY disorder of sex development

Steroid measurement is a challenge in pediatric endocrinology. Currently, liquid chromatography with tandem mass spectrometry (LC-MS/MS) is considered a gold standard for this purpose. The aim of this study was to compare both LC-MS/MS and immunoassay (IA) for androgens before and after human recombinant chorionic gonadotropin (rhCG) stimulus in children with 46,XY disorders of sex development (DSD). Nineteen patients with 46,XY DSD were evaluated; all of them were prepubertal and non-gonadectomized. Testosterone, dihydrotestosterone (DHT), DHEA and androstenedione were measured by IA and LC-MS/MS before and 7 days after rhCG injection. The correlation between IA and LC-MS/MS was analyzed by the intraclass correlation coefficient (ICC) and Spearman’s rank correlation coefficient (SCC). For concordance analysis the Passing and Bablok (PB) regression and the Bland and Altman (BA) method were used.Testosterone showed excellent correlation (ICC = 0.960 and SCC = 0.964); DHT showed insignificant and moderate correlations as indicated by ICC (0.222) and SCC (0.631), respectively; DHEA showed moderate correlation (ICC = 0.585 and SCC = 0.716); and androstenedione had poor and moderate correlations in ICC (0.363) and SCC (0.735), respectively. Using the PB method, all hormones showed a linear correlation, but proportional and systematic concordance errors were detected for androstenedione, systematic errors for testosterone and no errors for DHEA and DHT. By the BA method, there was a trend of IA to overestimate testosterone and androstenedione and underestimate DHEA and DHT when compared to LC-MS/MS.Traditional IA should be replaced by LC-MS/MS for the androgens measurement in prepubertal children whenever is possible911CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES472098/2011-0001FAEPEX (Process: 106/2014