Dissertations / Theses on the topic 'Precocious puberty'

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xvii, 181 p. : ill. Includes bibliographical references (p. 159-181). Available online via OhioLINK's ETD Center

AN ABSTRACT OF THE DISSERTATION OF LAN HAI, for the Doctor of Philosophy degree in Molecular Cellular and Systemic Physiology, presented on 11th December, 2015 at Southern Illinois University Carbondale. TITLE: EFFECT OF CONSTITUTIVELY ACTIVATED LUTEINIZING HORMONE RECEPTOR ON THE MOUSE REPRODUCTIVE SYSTEM MAJOR PROFESSOR: Dr. Prema Narayan The luteinizing hormone/chorionic gonadotropin receptor (LHCGR) is crucial for fertility, and genetic mutations in LHCGR cause adverse effects in reproductive development. Among the activating mutations identified in LHCGR, replacement of aspartic acid 578 by glycine (D578G) is the most common inherited mutation. Boys with this mutation undergo puberty by 2-4 years, caused by elevated testosterone in the context of prepubertal luteinizing hormone levels and present with Leydig cell hyperplasia. Clinically, these symptoms are associated with familial male-limited precocious puberty (FMPP). Our lab has published a mouse model for FMPP (KiLHRD582G) with D582G mutation equivalent to D578G in human LHCGR. We have previously demonstrated that KiLHRD582G male mice exhibited precocious puberty, Leydig cell hyperplasia and elevated testosterone and was a good model for FMPP. However, unlike women with the D578G mutation who show no abnormal phenotype, our studies revealed that female KiLHRD582G mice were infertile. KiLHRD582G female mice exhibit precocious puberty and irregular estrous cyclicity. A temporal study from 2-24 weeks of age indicated elevated steroid levels and upregulation of steroidogenic acute regulatory protein as well as several steroidogenic enzyme genes. Ovaries of KiLHRD582G mice exhibited significant pathology with the development of large hemorrhagic cysts as early as 3 weeks of age, extensive stromal cell hyperplasia with luteinization, numerous atretic follicles and granulosa cell tumors. Anovulation could not be rescued by exogenous gonadotropins. The body weights of the KiLHRD582G mice was higher that wild type counterparts, but there were no differences in the body fat composition. Hyperandrogenism and polycystic ovary phenotype was not accompanied by impaired glucose tolerance. Blocking the androgen action and estrogen synthesis indicated that reproductive phenotype was primarily due to excess estradiol. These studies demonstrate that activating LHCGR mutations do not produce the same phenotype in humans and mice and clearly illustrates species differences in the expression and regulation of LHCGR in the ovary. As we use male KiLHRD582G mice as breeders, we observed that the KiLHRD582G mice became progressive infertile, and only 8% of KiLHRD582G were fertile at 6 months of age despite normal sperm production. The infertile KiLHRD582G males were not able to form copulatory plugs in WT females, and mating studies suggested that the KiLHRD582G males were not capable of mating and/or ejaculating. Sexual behavioral testing revealed that the infertile KiLHRD582G males were capable of mounting the receptive WT females but were unable to achieve ejaculation indicating a problem with erectile and/or ejaculatory function. To address the reason for the ejaculatory dysfunction, we performed histopathological analysis of the accessory glands and penis. Hematoxylin and eosin staining showed that the normal columnar epithelium was replaced by pseudostratified columnar epithelium in the ampulla and several aggregates of chondrocyte metaplasia were apparent in the penile body of KiLHRD582G male mice. A temporal study indicated the histopathological changes in ampulla and penile body initiated at 7-8 and 12 weeks of age, respectively. Immunohistochemistry indicated that the chondrocytes stained positive for collagen type II, SOX9 and androgen receptor in the nucleus and for LHCGR in the cytoplasm. Penile fibrosis is a major cause of erectile dysfunction and is characterized by an increased collagen/smooth muscle ratio. However, our Image J analysis, hydroxyproline assay and western blot showed that KiLHRD582G penile body exhibited reduced levels of smooth muscle actin but similar total collagen content compared to WT mice. Thus, penile fibrosis was not responsible for the progressive infertility of adult KiLHRD582G mice. We also observed Leydig cell adenoma and disruption of spermatogenesis at 1 year of age. Our results suggest FMPP patients may be susceptible to infertility and testicular tumors later in their life and a follow-up study of FMPP patients is recommended.

IntroduÃÃo: A Puberdade Precoce (PP) à problema cada vez mais freqÃente em todo o mundo. CrianÃas em PP estÃo em risco de iniciaÃÃo sexual mais cedo, abuso sexual, baixa estatura na vida adulta, risco aumentado de obesidade, hiperinsulinemia e hipertensÃo arterial. O estudo trata da freqÃÃncia e condiÃÃes associadas à PP, visando propor medidas de SaÃde PÃblica para reduÃÃo deste problema e de suas possÃveis seqÃelas. MÃtodos: O estudo transversal, desenvolve abordagem analÃtica das crianÃas acometidas de PP, atendidas no ambulatÃrio de endocrinologia do Hospital UniversitÃrio Walter CantÃdio (HUWC) no perÃodo 1994 a 2010, contando com uma amostra de 342 crianÃas de 1 a 11 anos. As variÃveis analisadas incluÃram as caracterÃsticas biolÃgicas, sÃcioeconÃmicas, familiares e nutricionais. A anÃlise dos dados foi realizada atravÃs do programa SPSS,utilizando-se o teste do qui-quadrado para medir associaÃÃes, com o valor alfa de 5%. Resultados: Encontrou-se uma razÃo feminino: masculino de 37:1. As caracterÃsticas mais freqÃentes das crianÃas com PP foram: cor da pele parda, 64,1%; adotadas, 7,3%; sobrepeso e obesidade, 27,1 e 25,1%, respectivamente; inÃcio da puberdade abaixo dos 5 anos, 27,1%; altura dos pais abaixo da mÃdia populacional, 80%. CrianÃas com PP atendidas tardiamente pelo serviÃo especializado tenderam a apresentar mais cedo sinais da puberdade (p<0,001) e a pertencerem a famÃlias de menor renda (p<0,002). ConclusÃes: Encontraram-se elevadas proporÃÃes de crianÃas com PP que haviam sido adotadas e que apresentavam sobrepeso/obesidade, dois fatores de risco importantes na gÃnese do problema. O retardo na atenÃÃo especializada esteve associado ao aparecimento muito precoce dos sinais da puberdade e à baixa renda familiar, sugerindo a necessidade de aÃÃes de saÃde pÃblica que promovam a detecÃÃo e a atenÃÃo ao problema nas camadas mais pobres da populaÃÃo
Introduction: Precocious puberty (PP) is an increasingly common problem worldwide. PP children are at risk of early sexual initiation, sexual abuse, short stature in adult life, increased risk of obesity, hyperinsulinemia and hypertension. The study addresses the frequency and conditions associated with PP in order to propose measures to reduce this public health problem and its possible sequels. Methods: A cross-sectional study develops analytical approach to children affected by PP, who were given assistance at the clinic of Endocrinology, in the University Hospital Walter CantÃdio (HUWC) in the period from 1994 to 2010, with a sample of 342 children aged 1 to 11 years. The variables looked over included the biological, socio-economic, family and nutrition features. Data analysis was performed by using the SPSS program by means of the chi-square test to evaluate any association with the alpha value of 5%. Results: A ratio female-male of 37:1 was found. The most frequent features of children with PP were: dark skin, 64.1%; adopted, 7.3%; overweight and obesity, 27.1 and 25.1%, respectively; onset of puberty under 5 years, 27.1%; parental height below the average population, 80%. Children with late PP assistance by specialized service tended to show early signs of puberty (p <0.001) and belong to households with lower income (p <0.002). Conclusions: It was found a high proportion of children with PP which had been adopted and that had overweight/obesity, two important risk factors in the genesis of the problem. The delay in specialized care was associated with signs of very early onset of puberty and low household income, which suggests that public health actions are required to encourage detection and attention to the problem among the poorest strata of the population.

INTRODUÇÃO: Os fatores genéticos que influenciam o início da puberdade precoce ainda não são totalmente conhecidos. Assim, investigar os mecanismos gênicos que estariam envolvidos na sua gênese é muito importante, pois, além de possibilitar o diagnóstico em fases iniciais, pode contribuir para o desenvolvimento de novas terapias, com melhora do prognóstico. Para alguns investigadores, o estradiol também seria um fator contribuinte no determinismo da puberdade. OBJETIVOS: Estudar três genes que codificam enzimas relacionadas à esteroidogênese (CYP1A1, CYP1B1 e CYP17) em meninas com puberdade precoce central. Avaliar a associação entre variações na sequência desses genes e a puberdade precoce central. MÉTODOS: Foram incluídas 177 pacientes, divididas em dois grupos: Grupo Controle - formado por 104 meninas sem puberdade precoce, acompanhadas no Setor de Ginecologia da Infância e da Adolescência da Divisão de Clínica Ginecológica do HC-FMUSP por outros diagnósticos; Grupo Caso - composto por 73 meninas com diagnóstico de puberdade precoce central, acompanhadas no mesmo setor. Foi avaliada a presença de mutação em genes envolvidos no metabolismo do estrogênio (CYP1A1, CYP1B1 e CYP17) pela técnica de RFLP (Restriction Fragment Length Polymorphism), utilizando DNA obtido a partir de sangue periférico. RESULTADOS: A distribuição dos genótipos de CYP1A1 MspI (p=0,86) e CYP17 (p=0,12) não apresentou diferença significante entre os grupos. Para o CYP1B1 Eco571, o genótipo mutado C/C foi mais frequente no Grupo Controle que no Grupo Caso (p=0,03). CONCLUSÃO: Nossos dados sugerem que a variação do gene CYP1B1 Eco571 poderia estar associada ao determinismo da puberdade
INTRODUCTION: The genetic factors influencing onset of precocious puberty are not as yet fully known. Therefore, it is very important to investigate the genetic mechanisms involved in its genesis, for the resulting knowledge would not only enable diagnosis in the early stages but also contribute to the development of new therapies for improvement in prognosis. According to some researchers, estradiol would also be a contributory factor in puberty timing. OBJECTIVES: To investigate three genes which codify enzymes associated with steroidogenesis (CYP1A1, CYP1B1, and CYP17) in girls with central precocious puberty by focusing on the association between the sequence variation of these genes and central precocious puberty. METHODS: A total of 177 patients was included and divided into two groups: Control Group with 104 girls without precocious puberty who were being treated for other diagnoses at the Sector of Gynecology of Childhood and Adolescence, Division of Gynecology Clinic, HC-FMUSP; Case Group with 73 girls diagnosed with central precocious puberty. Mutations in genes involved in estrogen metabolism (CYP1A1, CYP1B1, and CYP17) were assessed by the RFLP (restriction fragment length polymorphism) technique using DNA obtained from peripheral blood. RESULTS: No significant difference in the distribution of the CYP1A1 MspI (p=0.86) and CYP17 (p=0.12) genotypes was detected between the two study groups. As for CYP1B1 Eco571, the mutated C/C genotype was found to be more frequent in the Control Group than in the Case Group (p=0.03). CONCLUSION: Our data suggest the CYP1B1 Eco571 gene variation is associated with puberty timing

Introdução - A puberdade precoce verdadeira ou dependente de GnRH apresenta importante morbidade: a baixa estatura, conseqüência da rápida progressão da idade óssea, além das seqüelas psico-emocionais do desenvolvimento sexual secundário precoce. Daí a importância da realização de um diagnóstico precoce e preciso, a fim de que a terapêutica adequada seja instituída o quanto antes. O uso do análogo do GnRH (aGnRH) em teste diagnóstico vem sendo utilizado com este objetivo. Neste estudo avaliou-se os valores de corte para o diagnóstico de puberdade precoce verdadeira, usando-se o teste do aGnRH. Material e métodos - Estudo prospectivo, com 44 meninas, com desenvolvimento dos caracteres sexuais secundários antes dos 8 anos de idade, atendidas no Ambulatório de Ginecologia Infanto-Puberal do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo. Realizou-se, em todos os casos, o teste do aGnRH, que consistiu na coleta de amostra sanguínea basal para dosagem de FSH e LH, seguida da aplicação subcutânea de 500µg de acetato de leuprolida (Lupron®). Novas amostras sanguíneas foram realizadas após 3 horas, para dosagem de FSH e LH, e após 24 horas da aplicação, para dosagem de estradiol Compararam-se os níveis de LH e FSH basais, de 3 horas e a relação LH/FSH obtida, além do estradiol de 24h, com a evolução clínica das pacientes. Este foi o padrão ouro utilizado para análise do teste, sendo que, após 6 meses, as pacientes foram divididas em 2 grupos: puberdade progressiva (puberdade precoce verdadeira) e não-progressiva. Para análise estatística, utilizou-se curvas ROC, estabelecendo-se sensibilidade, especificidade e melhor nível de corte para o diagnóstico de puberdade precoce verdadeira, para os diferentes critérios analisados. Além disso, avaliou-se a concordância entre os diversos tipos de análise do teste, através do coeficiente kappa. Resultados - O LH de 3 horas apresentou valor de corte > 4,5 mUI/mL, sensibilidade 59,1% e especificidade 86,4%, com área sobre a curva de 0,723. O valor de kappa foi de 0,45, com concordância de 0,73. O estradiol de 24 horas apresentou valor de corte > 40,6 pg/mL, sensibilidade 70% e especificidade 73,7%, com área sobre a curva de 0,703. O valor de kappa foi de 0,436, com concordância de 0,718. Dentre todos os critérios analisados, o melhor deles foi a relação LH/FSH de 3 horas, com valor de corte > 0,14, sensibilidade 72,7% e especificidade 77,3%, com área sobre a curva de 0,771. O valor de kappa foi de 0,5, com concordância de 0,75. Conclusões - Em nossa avaliação, a relação LH/FSH de 3 horas foi superior ao valor de LH de 3 horas ou estradiol de 24 horas, que haviam sidos os melhores critérios diagnósticos no trabalho pioneiro na utilização deste teste.
Introduction - True or GnRH-dependent precocious puberty involves important morbidity such as short stature due to the rapid progression of bone age, as well as psycho-emotional sequels of precocious secondary sexual development. Thus, it is important to make an early and precise diagnosis so that appropriate treatment can be instituted as early as possible. The GnRH analogue (aGnRH) in the diagnostic test has been used for this purpose. In the present study, the sensitivity and specificity of different laboratory criteria for the diagnosis of true precocious puberty were compared using the aGnRH test. Material and methods - This was a prospective study conducted on 44 girls with the development of secondary sexual traits before 8 years of age attended at the Childhood-Pubertal Gynecology Outpatient Clinic of the University Hospital, Faculty of Medicine of Ribeirão Preto, University of São Paulo. The aGnRH test was performed in all cases, consisting of collection of a basal blood sample for the determination of FSH and LH, followed by subcutaneous application of 500 µg leuprolide acetate (Lupron®). New blood samples were obtained after 3 hours, for the determination of FSH and LH, and after 24hours of application, for determination of estradiol. Basal LH and FSH levels and levels after 3 hours, the LH/FSH ratio obtained 3 hours after the administration of 500 µg Lupron®, and 24 hour estradiol levels were compared with the clinical course of the patients. This was the gold standard used for the analysis of the test and after 6 months the patients were divided into 2 groups: progressive puberty (true precocious puberty) and non-progressive puberty. ROC curves were used for statistical analysis, with the determination of the sensitivity, specificity and best cut-off value for the diagnosis of true precocious puberty of the different criteria analyzed. In addition, the agreement of the various types of test analysis was evaluated using the kappa coefficient. Results - Three hour LH presented a cut-off value of > 4.5 mIU/mL, 59.1% sensitivity and 86.4% specificity, with an area under the curve of 0.723. The kappa value was 0.45, with 0.73 agreement. Twenty-four hour estradiol presented a cut-off value of > 40.6 pg/mL, 70% sensitivity and 73.7% specificity, with an area under the curve of 0.703. The kappa value was 0.436, with 0.718 agreement. The best of all criteria used was the 3 hour LH/FSH ratio, with a cut-off value of > 0.14, 72.7% sensitivity and 77.3% specificity, with an area under the curve of 0.771. The kappa value was 0.5, with 0.75 agreement. Conclusions - In the present evaluation, the 3 hour LH/FSH ratio was superior to the 3 hour LH value and the 24 hour estradiol value, which had been the best diagnostic criteria in the pioneering study using this test.

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The objective of this study was to evaluate the prevalence of malocclusion, the need for orthodontic treatment and dentofacial changes in girls with Precocious Puberty (PP). This work gave rise to two distinct studies and data analyzes including descriptive statistics, Pearson's correlation analysis, paired t-test and t-test for one sample: 1- the cross-sectional study included 39 girls (6 to 11 years old) with confirmed PP diagnosis. The Dental Aesthetic Index (DAI) and the Index of Orthodontic Treatment Need (IOTN) were used for malocclusion evaluation and need for orthodontic treatment. Cephalometric analyzes were performed for the diagnosis of facial growth abnormalities. There was a high prevalence (64.1%) of severe and very severe malocclusion (DAI grades 3 and 4) and 82.1% of cases requiring moderate to high treatment (IOTN grades 3 and 4). 2- the control case study included 39 girls (6 to 11 years old) with confirmed PP diagnosis (case group). In this group, dental panoramic and hand and wrist radiographs were made. The control group consisted in 78 panoramic radiographs of girls without PP, randomly selected. Each panoramic radiograph of the 39 from case group was compared with two radiographs from control group, that is, a ratio of 1: 2. The chronological chart of dental mineralization of Nicodemus was used to identify the dental age. A significant difference (p <0.001) was found between chronology and bone age. Dental age (mean = 117) was lower than bone age (mean = 123) in girls with PP, but higher than dental age (mean = 111.5) in the control group. There was a significant correlation between chronological age and dental age. It was concluded that PP can have an impact on the development of dentition and craniofacial growth, a relevant factor for the diagnosis and the choice of the best moment for orthopedic / orthodontic intervention in malocclusion.
O objetivo deste estudo foi avaliar a prevalência de maloclusão, necessidade de tratamento ortodôntico e alterações dentofaciais em meninas com Puberdade Precoce (PP). Este trabalho deu origem a dois estudos distintos e as análises dos dados incluíram estatística descritiva, análise de correlação de Pearson, teste t pareado e teste t para uma amostra: 1- o estudo transversal incluiu 39 meninas (6 a 11 anos) com diagnóstico confirmado de PP. O Dental Aesthetic Index (DAI) e o Index of Orthodontic Treatment Need (IOTN) foram utilizados para a avaliação da maloclusão e necessidade de tratamento ortodôntico. Análises cefalométricas foram realizadas para o diagnóstico de anormalidades do crescimento facial. Foi observada alta prevalência (64,1%) de maloclusão grave e muito grave (DAI graus 3 e 4) e 82,1% dos casos com necessidade de tratamento moderada a alta (IOTN graus 3 e 4). 2- o estudo caso controle incluiu 39 meninas (6 a 11 anos) com diagnóstico confirmado de PP (grupo caso). Foram feitas, nesse grupo, radiografias panorâmicas e de mão e punho. O grupo controle foi composto por 78 radiografias panorâmicas de meninas sem PP, selecionadas aleatoriamente. Cada radiografia panorâmica das 39 do grupo caso foi comparada com duas radiografias do grupo controle, ou seja, uma razão de 1:2. A tabela cronologica de mineralização dental de Nicodemo foi utilizada para identificação da idade dentária. Foi encontrada uma diferença significativa (p<0,001) entre a idade cronologia e óssea. A idade dentária (média=117) foi menor que a idade óssea (média=123) nas meninas com PP, mas foi maior que a idade dentária (média= 111,5) do grupo controle. Houve correlação significativa entre a idade cronológica e a idade dentária. Concluiu-se que a PP pode ter impacto sobre o desenvolvimento da dentição e crescimento craniofacial, fator relevante para o diagnóstico e escolha do melhor momento para a intervenção ortopédica/ortodôntica da maloclusão

Idiopathic central precocious puberty may compromise adult height. Gonadotrophin-releasing hormone analogues (GnRHa) suppress puberty and increase final height prediction, but their influence on final height is unclear, especially in girls with pubertal onset just below eight years of age, the traditional limit of normal.
At seven children's hospitals, we identified 53 treated and 24 untreated patients (24 and 7 to final height, respectively) whose pubertal onset was between ages 5--8 years. At baseline, bone age advancement and predicted adult height were similar in the two groups. In both groups, the predicted adult height slightly overestimated the final height. What role, if any, GnRHa therapy played in preventing a shorter adult height is uncertain in these borderline cases. The substantial intra- and inter-observer variability in bone age readings compromised the utility of the height prediction method.
The methodological challenges inherent to this study are identified and discussed.

Avanços recentes na etiologia da puberdade precoce foram obtidos a partir da análise do genoma por sequenciamento global. Mutações inativadoras do gene MKRN3 representam uma causa importante de puberdade precoce central (PPC) familial (33-46% dos casos). O objetivo do estudo foi a análise do DNA genômico de pacientes com PPC de origem familial ou esporádica sem mutações deletérias no gene MKRN3. Foram selecionados 68 indivíduos com PPC (37 com a forma familial e 31, aparentemente, esporádicos). O DNA genômico foi extraído do sangue periférico ou da saliva dos pacientes com PPC. A técnica de sequenciamento genômico em larga escala (ILLUMNA -Clonal Single Molecule Array Technology - CSMA) foi usada na busca de novos genes implicados com o desenvolvimento puberal prematuro em seis indivíduos, sendo três afetados e três não afetados, pertencentes a uma grande família brasileira com PPC (Família 1). Mutações em um gene candidato foram pesquisadas em 64 pacientes por sequenciamento automático direto (método de Sanger). Em um subgrupo de pacientes, foi realizada a técnica de MLPA com sondas customizadas na busca de deleções. Por sequenciamento genômico global, foi identificado um novo complexo rearranjo no gene DLK1, caracterizado por uma deleção de, aproximadamente, 14.000 pb na região 5\' não traduzida (5\'UTR), englobando o início do exon 1, associada a uma duplicação de uma região do intron 3 de 269 pb. O gene DLK1 está localizado no braço longo do cromossomo 14 (14q32.2) e sofre imprinting materno. Este lócus está associado à síndrome de Temple, uma doença complexa com múltiplas manifestações, incluindo puberdade precoce central em até 90% dos casos. Para investigar o efeito dessa deleção genômica, as concentrações séricas da proteína DLK1 pelo método ELISA foram medidas nas pacientes afetadas da Família 1. Valores indetectáveis de DLK1 foram encontrados nestas pacientes. O fenótipo das pacientes afetadas da Família 1 caracterizou-se por uma PPC típica, sem sinais sindrômicos (excluída a síndrome de Temple). Posteriormente, por meio do sequenciamento direto, duas novas mutações inativadoras no gene DLK1 foram identificadas (p.Val272Cysfs*14 e p.Pro160Leufs*50) em duas famílias (Famílias 2 e 3) com PPC ou história de menarca precoce. O estudo de segregação nas Famílias 1 e 2 confirmou o padrão de herança autossômico dominante com penetrância completa e transmissão exclusiva pelo alelo paterno. A média de idade de início da puberdade nas pacientes afetadas do sexo feminino foi de 5,4 anos. A técnica de MLPA com sondas customizadas para o gene DLK1 não encontrou outras deleções no subgrupo estudado. Em conclusão, foram identificadas três mutações inativadoras no gene DLK1 associadas à PPC familial de origem paterna. O DLK1 é o segundo gene imprintado associado a distúrbios puberais em humanos. Este achado sugere um papel dos genes imprintados no controle da puberdade. O mecanismo pelo qual esse gene afeta a puberdade ainda é desconhecido
Recent advances in the etiology of precocious puberty were obtained from the whole-genome sequencing analysis. Inactivating mutations of the MKRN3 gene represent a major cause of familial central precocious puberty (CPP) (33%- 46% of the cases). The objective of the study was to analyze the genomic DNA of patients with familial or sporadic CPP without deleterious mutations in the MKRN3 gene. Sixty-eight individuals with CPP (37 with familial form and 31 apparently sporadic cases) were selected. The genomic DNA was extracted from the peripheral blood or saliva of patients with CPP. We used the whole-genomic sequencing technique (ILLUMNA - Clonal Single Molecule Array Technology - CSMA) searching for a new candidate genes implicated in premature pubertal development in 6 individuals, 3 affected and 3 non-affected, belonging to a large Brazilian family with CPP (Family 1). Mutations in one candidate gene were investigated in 64 patients through automatic sequencing (Sanger\'s method). In a subgroup of patients, MLPA using synthetic MLPA probes was performed to search for deletions. A new complex rearrangement in the DLK1 gene characterized by a deletion of approximately 14.000pb in the 5\' untranslated (5\'UTR), encompassing the start of exon 1, associated with a duplication of a region of intron 3 of 269 bp was identified by whole-genomic sequencing. The DLK1 gene is located on the long arm of chromosome 14 (14q32.2) and it is maternally imprinted gene. This locus is associated with Temple syndrome, a complex disorder with multiple alterations, including central precocious puberty in up to 90% of cases. To investigate the effect of this genomic deletion, a serum measurement of DKL1 protein using ELISA method was performed in the affected patients from Family 1. Undetectable serum DLK1 levels were found in these patients. The phenotype of affected patients from Family 1 was characterized by a typical CPP, without syndromic signs (excluding Temple syndrome). Posteriorly, two new inactivating mutations in the gene DLK1 were identified (p.Val272Cysfs*14 and p.Pro160Leufs*50) through direct sequencing in two families (Families 2 and 3) with CPP or precocious menarche history. The segregation studies in Families 1 and 2 confirmed the pattern of dominant autosomal inheritance with complete penetrance and exclusive transmission by the paternal allele. The average age of puberty onset in the affected female patients was 5.4 years. The MLPA technique with synthetic MLPA probes for the DLK1 gene did not find other deletions in the studied subgroup. In conclusion, we identified 3 paternally inherited inactivating mutations in the DLK1 gene associated with familial CPP. The DLK1 is the second imprinted gene associated with pubertal disorders in humans. This finding suggests a role of the imprinted genes in puberty control. The mechanism through which this gene affects puberty is still unknown

A idade normal para início da puberdade em meninas varia bastante, de 8 a 13 anos, e os genes envolvidos nesse controle são parcialmente conhecidos. Fatores ambientais, como alimentação e exposição a disruptores endócrinos, contribuem para essa variabilidade, de modo que genes modulados epigeneticamente podem justificar parte da complexidade desse processo. O termo epigenética se refere às modificações na expressão gênica que não são causadas por alterações na sequência do DNA. A metilação do DNA é o mecanismo epigenético mais bem estudado. Na última década surgiram evidências demonstrando a relação entre metilação do DNA e desenvolvimento puberal. Em fêmeas de roedores, a hipermetilação do DNA levou à puberdade precoce. Em humanos, a puberdade precoce central (PPC) familial causada por mutações nos genes MKRN3 e DLK1 é considerada um defeito do imprinting, fenômeno epigenético no qual apenas um dos alelos parentais é expresso, estando o outro metilado e inativo. Além disso, um conceito atual propõe que o início da puberdade requer a repressão epigenética de fatores inibidores do eixo gonadotrófico. Recentemente, genes zinc finger (ZNF) foram relacionados ao processo puberal, e muitos deles codificam repressores transcricionais. Neste trabalho, estudamos a metilação do DNA do sangue periférico de 10 pacientes do sexo feminino com PPC familial (casos índices) e 33 meninas com desenvolvimento puberal normal (15 pré-púberes e 18 púberes), usando a plataforma Human Methylation 450 BeadChip. Duas pacientes tinham PPC de causa genética (uma com mutação no MKRN3 e outra com deleção no DLK1) e oito tinham PPC idiopática, sem mutações identificadas pelo sequenciamento exômico global. Cento e vinte regiões diferencialmente metiladas foram identificadas entre as meninas saudáveis pré-púberes e púberes, estando 74% delas no cromossomo X. Apenas uma região mostrou-se hipometilada no grupo púbere e, de maneira importante, contém a região promotora do ZFP57, fator necessário para manutenção do imprinting. Uma vez que a hipermetilação nas regiões promotoras dos genes é relacionada à inibição transcricional, o achado de hipermetilação global do DNA na puberdade sugere que haja inibição de fatores inibidores do eixo gonadotrófico, o que resultaria no início do processo puberal. O receptor estrogênico destacou-se como um fator transcricional que se liga a sete genes diferencialmente metilados entre os controles pré-púberes e púberes. As pacientes com PPC apresentaram mais sítios CpG hipermetilados tanto na comparação com as meninas pré-púberes (81%) quanto púberes (89%). Há doze genes ZNF contendo sítios CpG hipermetilados na PPC. Não foram encontradas anormalidades de metilação nos genes MKRN3 e DLK1 nem em suas regiões regulatórias. Em conclusão, este estudo evidenciou hipermetilação global do DNA em meninas com puberdade normal e precoce, sugerindo que esse padrão é uma marca epigenética da puberdade. Pela primeira vez, mudanças no metiloma de pacientes com PPC foram descritas. Modificações na metilação de vários genes ZNF parecem compor a complexa rede de mecanismos que leva ao início da puberdade humana
Normal puberty initiation varies greatly among girls, from 8 to 13 years, and the genetic basis for its control is partially known. Environmental factors, such as nutrition and exposure to endocrine disruptors, contribute to this variance, and epigenetically modulated genes may justify some of the complexity observed in this process. Epigenetics refers to alterations in gene expression that are not caused by changes in DNA sequence itself. DNA methylation is the best studied epigenetic mechanism. In the last decade, evidence has emerged showing the relationship between DNA methylation and pubertal development. In female mice, DNA hypermethylation led to precocious puberty. In humans, familial central precocious puberty (CPP) caused by mutations in the MKRN3 and DLK1 genes is considered a disorder of imprinting, an epigenetic phenomenon in which only one parental allele is expressed, and the other allele is methylated and inactive. In addition, animal studies indicated that pubertal timing requires epigenetic repression of inhibitory factors of the gonadotrophic axis. Recently, zinc finger genes (ZNF) were related to pubertal development, many of which encode transcriptional repressors. In the present study, we analyzed the DNA methylation of peripheral blood samples from 10 female patients with familial CPP (index cases) and 33 girls with normal pubertal development (15 pre-pubertal and 18 pubertal), using the Human Methylation 450 BeadChip assay. Genetic CPP was diagnosed in two patients (one with a MKRN3 mutation and the other with a DLK1 deletion). The remaining eight cases with idiopathic CPP were previously evaluated by whole exome sequencing and no causative mutations were identified so far. We evidenced 120 differentially methylated regions between pre-pubertal and pubertal healthy girls, and 74% of them were located at the X chromosome. Only one genomic region was hypomethylated in the pubertal group. Of note, it contains the promoter region of ZFP57, an important factor for imprinting maintenance. As DNA hypermethylation in gene promoters is related to gene silencing, the finding of global DNA hypermethylation in puberty suggests inhibition of inhibitory factors of the hypothalamic-pituitary-gonadal axis that results in puberty onset. Importantly, the estrogen receptor was identified as a transcriptional factor that binds to seven differentially methylated genes associated with pubertal process. Patients with CPP exhibited more hypermethylated CpG sites compared to both pre-pubertal (81%) and pubertal (89%) controls. Twelve ZNF genes were recognized as having hypermethylated CpG sites in CPP. The methylation analyses of MKRN3 and DLK1 genes showed no abnormalities. In conclusion, this study revealed a widespread DNA hypermethylation in girls with normal and precocious puberty, suggesting that this pattern can be an epigenetic signature of puberty. For the first time, changes in the methylome of patients with CPP were described. We highlight that alterations in methylation levels of several ZNF genes may impact the onset of human puberty

Introdução: A puberdade é considerada precoce quando ocorre antes dos 8 anos nas meninas. É classificada como puberdade precoce central (PPC) quando decorre da ativação prematura do eixo gonadotrófico e é considerada idiopática quando não há alteração no sistema nervoso central. O bloqueio puberal com análogos de GnRH é o tratamento de escolha da PPC e visa a regressão ou estabilização dos caracteres sexuais secundários, desaceleração da velocidade de crescimento e da maturação óssea com melhora do prognóstico de estatura adulta e promoção do ajuste psicossocial da criança e dos familiares. Poucos estudos avaliaram o impacto psicológico da PPC e o benefício resultante do bloqueio puberal. Objetivos: (1) Comparar o escore de estresse entre pacientes com PPC idiopática antes e durante o bloqueio puberal com análogos de GnRH e um grupo controle. (2) Avaliar a dinâmica da personalidade por meio do teste projetivo HTP-F (house-tree-person-family) em meninas com PPC idiopática, antes e durante o bloqueio puberal com análogos de GnRH e um grupo controle. (3) Comparar a prevalência dos indicadores de personalidade obtidos no HTP-F entre pacientes com PPC idiopática antes e durante o bloqueio puberal com análogos de GnRH e um grupo controle. Pacientes e Métodos: As pacientes foram agrupadas em pré-tratamento (n=12) e em tratamento (n=22), sendo 11 do grupo pré-tratamento reavaliadas 1 ano após início do tratamento (corte longitudinal) e 11 avaliadas em uma ocasião durante o tratamento (corte transversal). O grupo controle foi constituído de 8 meninas com desenvolvimento puberal em idade adequada, pareadas por estadiamento puberal. A avaliação psicológica incluiu entrevista semiestruturada, questionário psicossocial, aplicação da Escala de Stress Infantil (ESI) e do teste projetivo House-Tree-Person-Family (HTP-F). Os resultados estão expressos em média e desvio padrão e comparados entre os grupos por métodos estatísticos apropriados. Resultados: No grupo pré-tratamento, 6/12 (50%) pacientes encontravam-se estressadas, 4/12 (33%) na fase de alerta e 2/12 (17%) na fase de resistência. No grupo em tratamento (braço longitudinal), 3/11 (27%) pacientes encontravam-se estressadas e na fase de alerta. No grupo em tratamento (corte transversal) foram verificadas que 4/11 (36%) pacientes encontravam-se estressadas e na fase de alerta. No grupo controle somente 2/8 (25%) encontraram-se estressadas e situadas na fase alerta. Houve diferença significativa da média de escore total de estresse entre o grupo pré-tratamento, comparados ao grupo longitudinal e ao grupo controle (p <0,05). No teste HTP-F, os indicadores psicológicos significativos no grupo pré-tratamento foram sentimento de introversão comparado ao grupo em tratamento (longitudinal), sexualidade aflorada comparado ao grupo em tratamento (transversal) e sentimento de inferioridade comparado ao grupo controle (p < 0,05). Os indicadores psicológicos prevalentes, embora sem significância estatística no grupo pré-tratamento foram: ansiedade, sexualidade aflorada, esquema corporal inadequado, interação social inadequada e sentimento de inferioridade comparado aos demais grupos. No grupo em tratamento (longitudinal) foram significativos traços depressivos (luto) e sentimento de extroversão comparado ao grupo pré-tratamento (p < 0,05). No grupo em tratamento (transversal) os indicadores psicológicos prevalentes foram aceitação da mudança corporal e traços agressivos comparados aos demais grupos, embora sem significância estatística. Conclusão: O bloqueio puberal apresentou impacto significativo na redução do escore de estresse em pacientes com PPC. A aplicação do HTP-F em meninas com PPC apontou indicadores valiosos na dinâmica da personalidade, e poderá ser de grande utilidade na avaliação inicial, bem como na intervenção de meninas com precocidade sexual
Introduction: Puberty is considered premature when it occurs before 8 years of age in girls. It is classified as central precocious puberty (CPP) when it is caused by premature activation of the gonadotropic axis and idiopathic when there is no change in the central nervous system. Pubertal block with GnRH analogues is the treatment of choice for CPP and aims to achieve regression or stabilization of secondary sexual characteristics, slowing growth velocity and bone maturation with improved adult height prognosis and promoting the child\'s and family\'s psychosocial adjustment. Few studies have assessed the psychological impact of CPP and the resulting benefits of pubertal block. Objectives: (1) Compare the stress score among patients with idiopathic CPP before and during pubertal block with GnRH analogues and a control group. (2) To assess personality dynamics through the projective HTP-F (house-tree-person-family) test in girls with idiopathic PPC, before and during pubertal block with GnRH analogues and in a control group. (3) To compare the prevalence of personality indicators obtained in the HTP-F test in patients with idiopathic CPP before and during pubertal block with GnRH analogues and a control group. Patients and Methods: Patients were grouped at pre-treatment (n = 12) and treatment (n = 22) groups, with 11 from the pretreatment group being reassessed 1 year after the start of treatment (longitudinal section) and 11 being assessed on one occasion during treatment (cross-section). The control group consisted of 8 girls with appropriate pubertal development for age, matched by pubertal stage. Psychological assessment included semi-structured interviews, psychosocial questionnaire, application of the Child Stress Scale (CSS) and the projective House-Tree-Person-Family (HTP-F) test. The results are expressed as mean and standard deviation and compared between groups through appropriate statistical methods. Results: In the pre-treatment group, 6/12 (50%) patients were stressed, 4/12 (33%) in the alert phase and 2/12 (17%) in the resistance phase. In the treatment group (longitudinal arm), 3/11 (27%) patients were stressed and in the alert phase. In the treatment group (cross-section), 4/11 (36%) patients were stressed and in the alert phase. In the control group only 2/8 (25%) were stressed and in the alert phase. Significant differences were observed in the mean total stress score of the pre-treatment group when compared to the longitudinal and the control groups (p < 0.05). In the HTP-F test, significant psychological indicators in the pre-treatment group were: feelings of introversion compared to the treatment group (longitudinal), emergence of sexuality, when compared to the treatment group (cross-sectional) and feelings of inferiority when compared to the control group (p < 0.05). The prevalent psychological indicators, although not statistically significant in the pre-treatment group were: anxiety, emergence of sexuality, inadequate body schema, inadequate social interaction and feelings of inferiority when compared to the other groups. In the treatment group (longitudinal) the following indicators were significant: depressive traits (bereavement) and feeling of extroversion, when compared to pre-treatment group (p < 0.05). In the treatment group (cross-sectional) the prevalent psychological indicators were acceptance of body changes and aggressive traits when compared to other groups, although not statistically significant. Conclusions: The pubertal block had significant impact on stress score reduction in patients with CPP. The use of the HTP-F test in girls with CPP disclosed valuable indicators in personality dynamics and can be very useful in the initial assessment and intervention of girls with sexual precocity

O mecanismo de controle da secreção de GnRH inclui diversas vias neuronais. Estudos em modelos animais identificaram genes que codificam fatores de transcrição, tais como TTF-1 (thyroid transcription factor 1) e EAP1 (enhanced at puberty), que atuam no controle transcricional de genes codificadores de fatores excitatórios (KiSS1 e GnRH) e inibitórios (preproencefalinas) regulando a secreção de GnRH. Em primatas, a expressão de EAP1 e TTF-1 aumenta, no início da puberdade, nas regiões hipotalâmicas envolvidas na secreção de GnRH. Nos modelos animais, a deleção pós-natal de TTF-1 e o silenciamento do EAP1 provocam atraso puberal e prejuízo na função reprodutiva. TTF-1 também está envolvido na morfogênese diencefálica, por meio da via de sinalização da família Sonic-Hedgehog. Anormalidades na secreção de GnRH resultam em distúrbios puberais, que variam de puberdade precoce central (PPC) a hipogonadismo hipogonadotrófico. Hipotetizamos que anormalidades genéticas no TTF-1 e EAP1 estejam envolvidas na patogênese dos distúrbios puberais centrais. A PPC pode ser idiopática ou devido a causas orgânicas, sendo o hamartoma hipotalâmico, uma malformação congênita não neoplásica, a mais conhecida. Os pacientes com PPC devido a hamartoma hipotalâmico podem cursar com alterações neurológicas e cognitivas. Nossos objetivos foram: estudar as regiões codificadora do TTF-1 e do EAP1 e a região promotora do TTF-1 em pacientes com distúrbios puberais centrais; estabelecer a prevalência, taxa de penetrância e modo de herança da forma familial de PPC e caracterizar as manifestações neurológicas e neurocognitivas de pacientes com PPC devido a hamartoma hipotalâmico. Foram selecionados 133 pacientes com distúrbios puberais centrais - PPC idiopática (n=71), PPC devido a hamartoma hipotalâmico (n=15) e hipogonadismo hipogonadotrópico isolado normósmico (HHIn) (n=47) - e controles (n=53). Os genes TTF-1 e EAP1 foram amplificados e submetidos a sequenciamento automático. Os tratos de poliglutamina e polialanina no EAP1 foram estudados por software de análise de tamanho de fragmento (GeneScan). A avaliação neurológica e neurocognitiva dos pacientes com PPC devido a hamartoma hipotalâmico consistiu de exame neurológico, eletroencefalograma, ressonância magnética de encéfalo e aplicação da escala de inteligência (WISC-III, WAIS-III, WPPSIR). Identificamos 25% de casos familiais de PPC, com modo de herança autossômica dominante e taxa de penetrância de 67,5%. Variantes alélicas no TTF-1 não foram identificadas nos pacientes estudados. No gene EAP1 foram identificadas quatro variantes alélicas sinônimas: p.E87E, p.A163A, p.Y415Y e uma nova variante alélica p.C758C, encontradas em pacientes com PPC e HHIn. A distribuição das frequências alélica e genotípica das variantes alélicas do EAP1 não diferiram entre pacientes com PPC, HHIn e controles (p >0,05). Nas regiões poliglutamina e polialanina 5 distal foi identificada variação similar no número de repetições glutamina e alanina em pacientes e controles. Não houve diferença significativa da frequência alélica em relação ao número de repetições glutamina e alanina entre os grupos PPC e HHIn (p >0,05). A avaliação neurológica dos pacientes com PPC devido a hamartoma hipotalâmico revelou epilepsia gelástica e crises focais com generalização em 3/15 (20%) pacientes. Não houve diferença significativa entre a mediana do maior diâmetro dos hamartomas dos pacientes com PPC com e sem epilepsia (13 e 10 mm, respectivamente). Quanto à forma, 10 hamartomas eram sésseis e 5 pedunculados, sendo que a forma pedunculada foi detectada exclusivamente em pacientes sem epilepsia. A avaliação neurocognitiva em 11 dos 15 pacientes com PPC devido a hamartoma hipotalâmico detectou 2 pacientes com epilepsia com QI significativamente menor que o grupo sem epilepsia (p <0,05). Em conclusão, (i) a considerável prevalência de casos familiais de PPC reforça a influência dos fatores genéticos na puberdade humana; (ii) mutações germinativas no TTF-1 e no EAP1 não estão envolvidas na patogênese dos distúrbios puberais centrais; (iii) a função neurocognitiva reduzida nos pacientes com hamartoma e epilepsia sugere um efeito deletério das crises convulsivas no sistema nervoso central
GnRH secretion control involves multiple neuronal pathways. Animal studies have identified genes which codifies transcription factors, such as TTF-1 (thyroid transcription factor 1) and EAP1 (enhanced at puberty), that act in the transcriptional control of genes that codifies excitatory (KiSS1 and GnRH) and inhibitory factors (preproenkephalines) regulating GnRH secretion. In nonhuman primates, expression of EAP1 and TTF-1 are increased at the hypothalamic regions involved in GnRH secretion, at the beginning of puberty. In animal models, post-natal TTF-1 deletion and silencing of EAP1 lead to pubertal delay and damage of reproductive function. TTF-1 is also involved in diencephalic morphogenesis, through signalization via Sonic-Hedgehog family. Abnormalities in GnRH secretion are responsible for pubertal disorders, varying from central precocious puberty (CPP) to hypogonadotropic hypogonadism. We hypothesized that genetic anomalies at TTF-1 and EAP1 are involved in the pathogenesis of central pubertal disorders. CPP may be idiopathic or due to organic alterations and hypothalamic hamartoma, a non-neoplasic congenital malformation, is the most frequent known organic cause. Patients with CPP due to hypothalamic hamartoma may have neurological and cognitive disfunctions. Our aims were: to evaluated the codifying region of TTF-1 and EAP1 and the promoter region of TTF-1 in patients with central pubertal disorders; to establish the prevalence, penetrance rate and inheritance mode of familial CPP and to characterize neurologic and neurocognitive aspects of patients with CPP due to hypothalamic hamartoma. We selected 133 patients with central pubertal disorders idiopathic CPP (n=71), CPP due to hypothalamic hamartoma (n=15) and normosmic isolated hypogonadropic hypogonadism (nIHH) (n=47) - and controls (n=53). TTF-1 and EAP1 genes were amplified and sequenced. Polyglutamine and polyalanine tracts of EAP1 were studied by a fragment size analyser software (GeneScan). Neurologic and neurocognitive evaluation of CPP patients due to hypothalamic hamartoma consisted of neurologic exam, electroencephalogram, brain magnetic resonance and application of intelligence scale (WISC-III, WAIS-III, WPPSI-R). We identified 25% of familial CPP cases with autosomal dominant mode of inheritance and penetrance rate of 67.5%. No TTF-1 allelic variants were identified in the patients analysed. At EAP1 gene, four synonimous allelic variants were identified: p.E87E, p.A163A, p.Y415Y and a new allelic variant p.C758C, found in CPP and nIHH patients. The allelic and genotypic distribution of theses variants of EAP1 did not differ among patients with CPP and nIHH, and controls (p >0.05). At polyglutamine and 5 distal polyalanine region, similar glutamine and alanine repeats variation was found. No significative difference of allelic frequency distribution regarding the number of glutamines and alanines repeats was found among the studied groups (p >0.05). Neurologic evaluation of CPP patients due to hypothalamic hamartoma revealed epilepsy and focal crisis with generalization in 3/15 (20%) of the patients. No significant difference between the median of the larger diameter of hypothalamic hamartoma of CPP patients with and without epilepsy was found (10 mm and 13 mm, respectively). Regarding the shape, 10 hamartomas were sessile and 5 pedunculated, and the pedunculated shape was found only in non epileptic patients. Neurocognitive evaluation performed in 11 of the 15 patients with CPP due to hypothalamic hamartoma detected 2 patients with epilepsy whose IQ were significantly lower than the IQ found in the group without epilepsy (p <0.05). In conclusion, (i) the considerable prevalence of familial CPP cases reinforce the influence of genetic factors in human puberty; (ii) germinative mutations in TTF-1 and EAP1 are not involved in the pathogenesis of central pubertal disorders; (iii) reduced neurocognitive function in patients with hypothalamic hamartoma and epilepsy suggests a deleterious effect of crisis at the central nervous system

O ácido gama-aminobutírico (GABA), principal neurotransmissor inibitório, está envolvido no mecanismo intrínseco do início da puberdade. Os efeitos inibitórios do GABA sobre a secreção de GnRH (hormônio hipotalâmico estimulador da secreção das gonadotrofinas) são mediados pelo receptor tipo A (GABAA) que é composto por diferentes subunidades organizadas de forma heteropentamérica. A subunidade a1, codificada pelo gene GABRA1 localizado no locus 5q34-35, é a mais implicada na atividade inibitória do GABA. A puberdade precoce dependente de gonadotrofinas (PPDG) predomina no sexo feminino, sendo na maioria dos casos idiopática. Recentemente, defeitos moleculares das subunidades dos receptores de GABA têm sido identificados em pacientes com anormalidades eletroencefalográficas específicas. Neste estudo, investigamos a presença de mutações funcionais ou polimorfismos do GABRA1 em meninas com a forma idiopática de PPDG e avaliamos as anormalidades EEG neste grupo. Trinta e uma meninas com diagnóstico clínico e hormonal da forma idiopática da PPDG, sendo 6 casos familiais (19,4%) e 25 casos esporádicos (80,6%), e 73 controles não relacionados foram selecionados. Todas as pacientes com PPDG apresentaram ressonância magnética de sistema nervoso central normal. Vinte e três meninas foram submetidas a estudo eletroencefalográfico (EEG). O DNA genômico foi extraído do sangue periférico de todas as pacientes e controles. A região codificadora do GABRA1 foi amplificada utilizando-se oligonucleotídeos intrônicos específicos, seguida por purificação enzimática e seqüenciamento automático. Dois polimorfismos conhecidos do GABRA1 foram também estudados pelo programa GeneScan e pela técnica de digestão enzimática (enzima TaiI). O seqüenciamento automático do GABRA1 não revelou mutações funcionais. Identificamos 7 polimorfismos no GABRA1: duas variantes exônicas 156T>C e 1323G>A, localizados no éxons 4 e 11, respectivamente, e 5 polimorfismos intrônicos - IVS2-712(GT)n, no íntron 2, caracterizado por número variável de repetições GT; IVS3+12A>T, no íntron 3; IVS8+45T>G no íntron 8; IVS9+76T>G no íntron 9 e IVS10+15G>A, no íntron 10. Estes polimorfismos não alteram o uso do sítio de splice original. Não houve diferença estatisticamente significante entre a distribuição genotípica e a freqüência alélica dos 2 polimorfismos exônicos e do polimorfismo IVS2-712(GT) encontrados no grupo de pacientes e no grupo controle. O EEG revelou anormalidades em 6 de 23 meninas (4 sem epilepsia). A distribuição genotípica e a freqüência alélica dos polimorfismos do GABRA1 não difereriram significativamente entre as pacientes com PPDG sem e com anormalidades eletroencefalográficas. Nós concluímos que mutações funcionais ou polimorfismos no GABRA1 não estão envolvidos na etiologia da forma idiopática da PPDG e não estão associadas às anormalidades eletroencefalográficas encontradas. Adicionalmente, a presença de alterações eletroencefalográficas em pacientes com PPDG sem epilepsia sugere que a análise eletroencefalográfica deva ser incluída na investigação da PPDG
The gamma-aminobutyric acid (GABA), a dominant inhibitory neurotransmitter, is involved in the intrinsic mechanism of the onset of the puberty. Their inhibitory effects on the GnRH (hypothalamic gonadotropin release hormone) secretion are mediated by type A receptor (GABAA), composed by different subunits which are organized in a heteropentameric form. The alpha-1 subunit, encode by GABRA1 gene located at locus 5q34- 35, is the most implicated in the inhibitory activity of GABA. The gonadotropin-dependent precocious puberty (GDPP) is predominant in females, being idiopathic in the majority of the cases. Recently, molecular defects of the GABA receptor subunits have been identified in patients with specific electroencephalographic (EEG) abnormalities. In this study, we investigated the presence of functional mutations or polymorphisms of the GABRA1 in girls with the idiopathic form of the GDPP and evaluated EEG abnormalities in this group. Thirty-one girls with clinical and hormonal diagnosis of GDPP idiopathic form, being 6 familial cases (19.4%) and 25 sporadic cases (80.6%), and 73 unrelated controls were selected. All patients with GDPP had normal magnetic resonance of central nervous system. Twenty-three girls were submitted to electroencephalographic study. Genomic DNA was extracted from peripheral blood of all patients and controls. The entire coding region of the GABRA1 was amplified using specific intronic oligonucleotides, followed by enzymatic purification and automatic sequencing. Two known polymorphisms of the GABRA1 were also studied by GeneScan software and digestion with restriction endonuclease TaiI. The automatic sequencing of the GABRA1 did not reveal any functional mutations. We identified 7 polymorphisms in the GABRA1: two silent exonic variants 156T>C e 1323G>A, located at exons 4 e 11, respectively, e 5 polymorphisms - IVS2-712(GT)n, at intron 2, characterized by a variable number of repeat GT; IVS3+12A>T, at intron 3; IVS8+45T>G at intron 8; IVS9+76T>G at intron 9 and IVS10+15G>A, at íntron 10. These polymorphisms did not alter the use of original splicing site. No significant statistical difference of the genotypic distribution and allele frequency of the exonic polymorphisms (156T>C and 1323G>A) and IVS2-712(GT)n between unrelated patients and control group was obtained. Electroencephalographic tracings were abnormal in 6 of 23 girls (4 without epilepsy). No significant statistical difference of the genotype distribution and allele frequence were found between patients without and with EEG abnormalities. We conclude that functional mutations or polymorphisms in the GABRA1 are not involved in the etiology of idiopathic GDPP in this study, and they are not associated with electroencephalografic abnormalities. In addition, EEG abnormalities present in girls with GDPP without epilepsy, suggest that EEG analysis should be included in the investigation of the precocious puberty.

A puberdade é um processo biológico complexo do desenvolvimento sexual que tem início no final da infância e se caracteriza pela maturação do eixo hipotálamo-hipófise-gonadal, pelo desenvolvimento dos caracteres sexuais secundários, aceleração do crescimento e finalmente pela capacidade reprodutiva. Nos últimos anos, o peptídeo kisspeptina e seu receptor KISS1R têm sido fortemente envolvidos na regulação da secreção pulsátil do GnRH hipotalâmico e, portanto, com o início da puberdade humana. Mutações nos genes KISS1R e KISS1 foram identificadas em crianças brasileiras com puberdade precoce central (PPC). Estudos em famílias e em irmãs gêmeas estimaram que 50-70% da variação na idade de menarca pode ser hereditária, porém, até pouco tempo atrás, não se tinha conhecimento de variantes genéticas comuns que influenciassem no tempo de puberdade. Recentemente, quatro estudos independentes de associação ampla do genoma estabeleceram que marcadores genéticos próximos ou dentro do gene LIN28B estavam relacionados com a idade da menarca em mulheres normais. Além disso, mutações recessivas no gene lin28 levaram ao desenvolvimento precoce no C. elegans. Camundongos que superexpressam Lin28a apresentaram um retardo no desenvolvimento sexual. Com base nesses achados, investigamos a presença de variantes conhecidas ou novas nos genes KISS1, KISS1R e LIN28B em um grupo de crianças portadoras de PPC idiopática com o intuito de estabelecermos a prevalência dessas mutações na etiologia do desenvolvimento sexual prematuro em humanos. Cento e sete crianças com PPC (101 meninas e 6 meninos) foram selecionados, incluindo casos esporádicos e familiares. A população controle consistiu de 200 indivíduos adultos com história de desenvolvimento puberal normal em idade apropriada. A região promotora e os três exons do gene KISS1, os cinco exons do gene K1SS1R e os quatro exons do gene LIN28B foram amplificados e submetidos à sequenciamento automático. Uma variante em homozigose no gene KISS1, descrita anteriormente por pesquisadores do nosso laboratório, p.H90D, foi identificada em mais três crianças não relacionadas, portadoras de PPC idiopática. Essa variante está localizada no exon 3 do KISS1, levando a substituição de uma histidina por um ácido aspártico na posição 90 da kisspeptina-1 (p.H90D), correspondendo à região amino-terminal da kisspeptina-54 e estava ausente em 200 controles brasileiros. Estudos prévios in vitro com a variante p.H90D não revelaram alterações na capacidade de ligação ou ativação do KISS1R e na resistência a degradação. As mutações ativadoras p.R386P do KISS1R e p.P74S da kisspeptina, previamente descritas em puberdade precoce central, não foram identificadas no estudo atual. Uma nova e rara variante em heterozigose no gene LIN28B, p.H199R, foi identificada em uma menina brasileira com PPC idiopática. Essa variante está localizada no exon 4 do LIN28B, levando a substituição de uma histidina conservada por uma arginina na posição 199 da proteína (p.H199R) e estava ausente em 200 controles brasileiros. O pai da paciente, que apresentou desenvolvimento puberal normal, era portador da mesma variante em heterozigose. Estudos in vitro revelaram que a variante p.H199R não afeta a função de LIN28B na regulação da expressão do miRNA let-7. Outra variante alélica no gene LIN28B foi identificada numa menina com PPC. Essa variante estava localizada no íntron 2 do gene e uma análise computacional demonstrou que ela não altera o sítio de splicing no RNA maduro. Em conclusão, observamos que mutações nos genes KISS1 e KISS1R têm uma baixa prevalência em crianças com puberdade precoce central idiopática. Descrevemos uma nova e rara variante no gene LIN28B (p.H199R) numa menina com puberdade precoce central e os estudos funcionais do LIN28B selvagem ou contendo a variante p.H199R sugeriram que essa variante não está relacionada ao fenótipo de puberdade precoce
Puberty is a complex biological process of sexual development that begins in the late childhood and it is characterized by the maturation of the hipothalamic-pituitary-gonadal axis, secondary sexual characteristics development, growth acceleration and acquisition of the reproductive capacity. Over the last years, the kisspeptin peptide and its receptor KISS1R have been envolved in the regulation of the pulsatile hipothalamic GnRH secretion and consequently with the beginning of the puberty human. Researchers from our laboratory identified mutations in the KISS1R and KISS1 genes in Brazilian children with central precocious puberty (CPP). Studies performed in families and twins estimated that 50%-70% of the variation in the menarce age can be hereditary, however, until last years, we did not have knowledgment of the influence of commun genetic variants in the puberty time. Recently, four independent Genome-Wide Association Studies established that genetic markers near or inside of LIN28B gene were related with the menarce age in normal women. Furthermore, recessive mutations in the LIN28B gene caused a precocious develpment in C. elegans. Interestingly, mouse that overexpress Lin28a exhibited a sexual development delay. Accordingly with these datas investigated the presence of known or new variants in the KISS1, KISS1R and LIN28B genes in a larger cohort of children with CPP to establish the prevalence of these mutations in the etiology of premature sexual development in humans. 107 children with CPP (101 girls and 6 boys) were selected, including sporadic and familial cases. The control population consisted of 200 adults with normal pubertal development. The promoter region and the three exons of KISS1 gene, five exons of KISS1R and four exons of LIN28B were amplified and automatically sequenced. A homozygous variant previously described by researchers from our laboratory in the KISS1 gene, p.H90D, was identified in more 3 no related children with CPP idiophatic. This variant is located in exon 3 of KISS1, resulting in substitution of a histidine to an aspartic acid at position 90 of kisspeptin-1 (p.H90D), in the amino-terminal region of the protein-54 and was absent in 200 Brazilian controls. Previous studies in vitro with the p.H90D variant did not show alterations in the binding or activation capacity and in the resistance to degradation. The activating mutations p.R386P of the KISS1R and p.P74S of the kisspeptin, previously described in central precocious puberty, were not identified in the present study. A new and rare heterozygous variant in the LIN28B gene, p.H199R, was identified in a Brazilian girl with CPP idiophatic. This variant is located in exon 4 of the LIN28B, resulting in substitution of a histidine to an arginine at position 199 of protein (p.H199R) and was absent in 200 Brazilian controls. Her father, which had normal pubertal development, carried the same heterozygous variant. Studies in vitro revealed p.H199R did not affect the function of Lin28B in the regulation of let-7 miRNA expression. Another allelic variant in the LIN28B gene was identified in a girl with CPP. This variant was located in intron 2 of the gene and an in silico analysis showed that it does not change the splicing site in mature RNA. In conclusion, we observed that mutations in the KISS1 and KISS1R genes have a low prevalence in children with idiopathic central precocious puberty. We described a new and rare variant in LIN28B gene (p.H199R) in a girl with central precocious puberty and functional studies of the wild LIN28B or containing p.H199R variant suggested that p.H199R variant of the LIN28B is not related to the precocious puberty phenotype

Mutações inativadoras nos genes TAC3 e TACR3, os quais codificam a neurocinina B (NKB) e o seu receptor NK3R, respectivamente, foram descritas em pacientes com hipogonadismo hipogonadotrófico isolado (HHI) normósmico. A partir desse achado, hipotetizamos que mutações ativadoras na NKB e/ou NK3R resultariam na secreção prematura de GnRH e, consequentemente, no desenvolvimento de puberdade precoce dependente de gonadotrofinas (PPDG). Nesse estudo, investigamos a presença de mutações ativadoras e/ou polimorfismos nos genes TAC3 e TACR3 em pacientes com PPDG, bem como mutações inativadoras e/ou polimorfismos nesses genes em pacientes com retardo constitucional do crescimento e desenvolvimento (RCCD), e HHI normósmico. Duzentos e trinta e sete pacientes com distúrbios puberais centrais idiopáticos foram selecionados, sendo 114 com PPDG, 50 com RCCD, e 73 com HHI normósmico. Um grupo de 150 indivíduos que apresentaram desenvolvimento puberal normal foi utilizado como controle. As regiões codificadoras dos genes TAC3 e TACR3 foram amplificadas pela reação em cadeia da polimerase, seguido de purificação enzimática e seqüenciamento automático direto. Análises in silico e in vitro foram realizadas. Um nova variante foi identificada no gene TAC3, p.A63P, em uma paciente do sexo feminino com PPDG, a qual desenvolveu puberdade aos sete anos de idade. Essa variante (p.A63P) está localizada na proneurocinina B, e análises in silico sugeriram que ela não altera sítios constitutivos de splicing e é benigna para a estrutura da proteína. A análise de segregação familiar mostrou que a mãe da paciente, a qual apresentou um desenvolvimento puberal normal, também apresentava a alteração p.A63P em heterozigose, sugerindo que essa variante não desempenha um papel direto no fenótipo de PPDG. Uma nova variante em heterozigose no gene TACR3, p.A449S, foi identificada em uma paciente do sexo feminino com RCCD, que teve início puberal aos treze anos de idade. A análise do grau de conservação da alanina na posição 449 mostrou que esse aminoácido não é conservado entre as diferentes espécies, e análises in silico sugeriram que essa variante não altera os sítios constitutivos de splicing, e é benigna para a estrutura do NK3R. Três novas variantes no NK3R foram identificadas, p.G18D, p.L58L (c.172C>T) e p.W275*, em três pacientes do sexo masculino não relacionados com HHI normósmico. As variantes p.G18D e p.L58L foram identificadas em heterozigose, enquanto que a variante p.W275* foi identificada em heterozigose associada a variante silenciosa p.L58L (c.172C>T), e em homozigose em outro paciente. Análises in silico sugeriram que a variante p.G18D poderia afetar a funcionalidade do NK3R. Estudos in vitro dessa nova variante foram realizados, e mostraram que a mesma não altera a função do NK3R, visto que o aumento na produção de fosfatidil inositol não diferiu significativamente entre o receptor mutado e selvagem. Todas as novas variantes descritas nos genes TAC3 e TACR3 não foram identificadas em 300 alelos controles. Em conclusão, nosso trabalho identificou novas variantes nos genes TAC3 e TACR3 em pacientes brasileiros com distúrbios puberais centrais idiopáticos, e confirmou o envolvimento do complexo NKB/NK3R na etiologia do HHI normósmico
Inactivating mutations of the TAC3 and TACR3 genes, which encode the neurokinin B (NKB) and its receptor, NK3R, respectively, were described in patients with normosmic isolated hypogonadotropic hypogonadism (IHH). Based on these observations, we hypothesized that gain-of-function mutations in the NKB and/or NK3R might be associated with premature activation of GnRH release, leading to gonadotropin-dependent precocious puberty (GDPP). In this study, we investigated the presence of activating mutations and/or polymorphisms in the TAC3 and TACR3 genes in patients with GDPP, and inactivating mutations and/or polymorphisms in these genes in patients with constitutional delay of growth and puberty (CDGP) and normosmic IHH. It was selected 237 patients with idiopathic central pubertal disorders: 114 with GDPP, 50 with CDGP, and 73 with normosmic IHH. Indeed, a group 150 individuals who had puberty at adequate age was used as controls. The coding regions of TAC3 and TACR3 genes were amplified by polymerase chain reaction followed by enzymatic purification and direct automatic sequencing. In silico and in vitro analyses were performed. A new heterozygous variant in the TAC3 gene, p.A63P, was identified in a Brazilian girl with GDPP who had puberty onset at seven years of age. The p.A63P variant was located in the proneurokinin B and in silico analysis suggested that this variant does not alter constitutive splice sites, and it was benign to the protein. The segregation analysis revealed that her mother was heterozygous for the p.A63P variant (who had a normal pubertal development), suggesting that this variant does not play a role in the GDPP phenotype. It was identified a new heterozygous variant, p.A449S, in the TACR3 gene in a Brazilian girl with CDGP, who had puberty onset at thirteen years of age. Conservation degree analysis of alanine at position 449 showed that this amino acid is not a conserved residue among different species. In silico analyses suggested that this new variant does not alter splice sites or affects the structure of NK3R. Indeed, it was identified three new distinct variants in the TACR3 gene, p.G18D, p.L58L (c.172C>T) and p.W275*, in three unrelated males with normosmic IHH. Both p.G18D and p.L58L (c.172C>T) were identified in heterozygous state, and the p.W275* variant was identified in two of these males, since one in homozygous and in another in heterozygous state in association with the silent variant p.L58L (c.172C>T). In silico analyses suggested that p.G18D might be damaging to the NK3R. In vitro studies of this variant (p.G18D) showed that the amount of inositol phosphate (IP) was not significantly different in cells transfected with the p.G18D mutant receptor than in cells transfected with the wild type receptor, indicating that this variant did not alter the function of the neurokinin B receptor. All new variants identified in the TAC3 and TACR3 genes were absent in 300 control alleles. In conclusion, we identified new variants in the TAC3 and TACR3 genes in Brazilian patients with idiopathic central pubertal disorders. We confirm the key role of the NKB/NK3R complex in the etiology of normosmic IHH

O complexo de sinalização kisspeptina-GPR54 é um regulador chave para ativação dos neurônios de GnRH e do eixo reprodutivo. Mutações inativadoras no GPR54 foram identificadas em pacientes com hipogonadismo hipogonadotrófico normósmico isolado (HHIn). A partir desse achado, hipotetizamos que mutações ativadoras no GPR54 resultariam na liberação prematura de GnRH e, conseqüentemente, no aparecimento de puberdade precoce, dependente de gonadotrofinas (PPDG). No presente estudo, investigamos a presença de mutações ativadoras e/ou polimorfismos em pacientes com PPDG, assim como a presença de mutações inativadoras e/ou polimorfismos em pacientes HHIn ou retardo constitucional do crescimento e desenvolvimento puberal (RCCP). Cento e catorze pacientes com distúrbios puberais centrais idiopáticos foram selecionados, sendo 53 com PPDG, 33 com HHIn e 28 com RCCP. Cento e cinqüenta controles brasileiros que relatavam desenvolvimento puberal normal foram estudados. A região codificadora do GPR54 de todos os pacientes foi amplificada utilizando-se oligonucleotídeos intrônicos específicos, seguida de purificação enzimática e seqüenciamento automático. No grupo de puberdade precoce, identificamos uma nova variante em heterozigose no exon 5 do GPR54, que se caracterizou pela troca do aminoácido arginina por prolina na posição 386 (R386P) do receptor. Esta substituição foi encontrada em uma menina adotada com PPDG e estava ausente nos controles normais. Estudos in vitro demonstraram que as quantidades de fosfatidil-inositol (IP) e o grau de fosforilação da quinase regulada por sinal extracelular (pERK) em condições basais não foram significativamente diferentes entre as células transfectadas com o receptor selvagem ou com o receptor contendo a mutação R386P, indicando que não havia ativação constitutiva do receptor. No entanto, estudos por tempos mais prolongados demonstraram que a quantidade de IP e o grau de pERK permaneceram significativamente mais altos nas células transfectadas com o receptor mutante quando comparadas ao selvagem, indicando ativação da sinalização intracelular, porém por um mecanismo não-constitutivo. No grupo de hipogonadismo, duas novas variantes foram identificadas em três pacientes. Uma mutação do tipo inserção/deleção (indel) em homozigoze no sítio aceptor de splicing no intron 2 (IVS2-4_-2delGCAinsACCGGCT) do GPR54 foi identificada em dois irmãos com HHIn. Uma troca em heterozigose, E252Q, foi identificada em um paciente com HHIn esporádico. As duas alterações estavam ausentes no grupo controle. Polimorfismos foram encontrados nos pacientes com RCCP. Em conclusão, descrevemos a primeira mutação ativadora do GPR54 associada ao fenótipo de PPDG. Descrevemos uma nova mutação inativadora em sítio de splicing em pacientes com HHIn, entretanto mutações inativadoras do GPR54 são uma causa rara de HHIn.
The kisspeptin-GPR54 signaling complex is a gatekeeper of pubertal activation of GnRH neurons and of the reproductive axis. Inactivating mutations in the GPR54 receptor were identified in patients with normosmic isolated hypogonadotropic hypogonadism (nIHH). Based on this observation, we hypothesized that gain-of-function mutations of the human GPR54 receptor might be associated with premature activation of GnRH release, leading to gonadotropin-dependent precocious puberty (GDPP). In the present study, we investigated the presence of GPR54 activating mutations or polymorphisms in patients with GDPP and inactivating mutations or polymorphisms in patients with nIHH or constitucional delay of puberty (CDP). A hundred fourteen patients were selected; 53 with GDPP, 33 with nIHH and 28 with CDP. A hundred and fifty Brazilian controls who reported normal pubertal development were also studied. The entire coding region of GPR54 of all patients was amplified using specific intronic oligonucleotides followed by enzymatic purification and automated sequencing. We have identified a novel variant in heterozygous state in exon 5 of GPR54, R386P, in an adopted girl with GDPP. This substitution was absent in all controls. Basal inositol phosphate (IP) and phosphorilated extracellular signalregulated kinase (pERK) levels in cells transfected with WT or R386P GPR54 were not significantly different indicating that there was not a constitutive activation of the receptor. However, studies performed in more prolonged times demonstrated that the IP and the pERK levels were significantly higher in cells transfected with the mutant receptor when compared to the wild type, indicating that the signaling pathway was still activated although by a non-constitutive mechanism. In the nIHH cohort, we have identified two novel variants in three patients. The first variant was an insertion/deletion (indel) in homozygous state within the constitutive acceptor splice site of intron 2 of GPR54 (IVS2-4_-2delGCAinsACCGGCT) identified in two male siblings with nIHH. The second variant was the change E252Q in heterozygous state in a patient with sporadic nIHH. Both alterations were absent in the control population. We have found only polymorphisms in patients with CDP. In conclusion, we have described the first activating mutation in GPR54 associated with the GDPP phenotype. We have also described a novel splice site inactivating mutation in patients with nIHH however, inactivating mutations of GPR54 represent a rare cause of nIHH.

A maioria dos casos de puberdade precoce central (PPC) em meninas permanece idiopática. A hipótese de uma causa genética vem se fortalecendo após a descoberta de alguns genes associados a este fenótipo, sobretudo aqueles implicados com o sistema kisspeptina (KISS1 e KISS1R). Entretanto, apenas casos isolados de PPC foram relacionados à mutação na kisspeptina ou em seu receptor. Até recentemente, a maioria dos estudos genéticos em PPC buscava genes candidatos selecionados com base em modelos animais, análise genética de pacientes com hipogonadismo hipogonadotrófico, ou ainda, nos estudos de associação ampla do genoma. Neste trabalho, foi utilizado o sequenciamento exômico global, uma metodologia mais moderna de sequenciamento, para identificar variantes associadas ao fenótipo de PPC. Trinta e seis indivíduos com a forma de PPC familial (19 famílias) e 213 casos aparentemente esporádicos foram inicialmente selecionados. A forma familial foi definida pela presença de mais de um membro afetado na família. DNA genômico foi extraído dos leucócitos do sangue periférico de todos os pacientes. O estudo de sequenciamento exômico global realizado pela técnica ILLUMINA, em 40 membros de 15 famílias com PPC, identificou mutações inativadoras em um único gene, MKRN3, em cinco dessas famílias. Pesquisa de mutação no MKRN3 realizada por sequenciamento direto em duas famílias adicionais (quatro pacientes) identificou duas novas variantes nesse gene. O MKRN3 é um gene de um único éxon, localizado no cromossomo 15 em uma região crítica para a síndrome de Prader Willi. O gene MKRN3 sofre imprinting materno, sendo expresso apenas pelo alelo paterno. A descoberta de mutações em pacientes com PPC familial despertou o interesse para a pesquisa de mutações nesse gene em 213 pacientes com PPC aparentemente esporádica por meio de reação em cadeia de polimerase seguida de purificação enzimática e sequenciamento automático direto (Sanger). Três novas mutações e duas já anteriormente identificadas, incluindo quatro frameshifts e uma variante missense, foram encontradas, em heterozigose, em seis meninas não relacionadas. Todas as novas variantes identificadas estavam ausentes nos bancos de dados (1000 Genomes e Exome Variant Server). O estudo de segregação familial em três dessas meninas com PPC aparentemente esporádica e mutação no MKRN3 confirmou o padrão de herança autossômica dominante com penetrância completa e transmissão exclusiva pelo alelo paterno, demonstrando que esses casos eram, na verdade, também familiares. A maioria das mutações encontradas no MKRN3 era do tipo frameshift ou nonsense, levando a stop códons prematuros e proteínas truncadas e, portanto, confirmando a associação com o fenótipo. As duas mutações missenses (p.Arg365Ser e p.Phe417Ile) identificadas estavam localizadas em regiões de dedo ou anel de zinco, importantes para a função da proteína. Além disso, os estudos in silico dessas duas variantes demonstraram patogenicidade. Todos os pacientes com mutação no MKRN3 apresentavam características clínicas e hormonais típicas de ativação prematura do eixo reprodutivo. A mediana de idade de início da puberdade foi de 6 anos nas meninas (variando de 3 a 6,5) e 8 anos nos meninos (variando de 5,9 a 8,5). Tendo em vista o fenômeno de imprinting, análise de metilação foi também realizada em um subgrupo de 52 pacientes com PPC pela técnica de MS-MLPA, mas não foram encontradas alterações no padrão de metilação. Em conclusão, este trabalho identificou um novo gene associado ao fenótipo de PPC. Atualmente, mutações inativadoras no MKRN3 representam a causa genética mais comum de PPC familial (33%). O MKRN3 é o primeiro gene imprintado associado a distúrbios puberais em humanos. O mecanismo preciso de ação desse gene na regulação da secreção de GnRH necessita de estudos adicionais
Most cases of central precocious puberty (CPP) in girls remain idiopathic. The hypothesis of a genetic cause has been strengthened after the discovery of some genes associated with this phenotype, particularly those involved with the kisspeptin system (KISS1 and KISS1R). However, genetic defects in KISS1 and its receptor are rare and have been identified in only a few patients with CPP.over the past years. To date, most genetic studies in CPP was based mainly on a candidate gene approach, including genes selected in animal studies, human models of patients with hypogonadotropic hypogonadism or in genome wide association studies. In the present study, we used whole exome sequencing, a more advanced method of sequencing, to identify variants associated with CPP. Thirty-six patients with the familial form of CPP (19 families) and 213 apparently sporadic cases were initially selected. The familial form was defined by the presence of more than one member affected in the family. Genomic DNA was extracted from peripheral blood leukocytes in all patients. Whole exome sequencing performed by ILLUMINA technique in 40 members of 15 families with CPP, identified inactivating mutations in a single gene, MKRN3, in five out of these families. Analysis of MKRN3 mutations performed by automatic sequencing in two additional families (four patients) identified two novel mutations. MKRN3 is an introless gene located on chromosome 15, in the Prader Willi syndrome critical region, and it is expressed only by the paternal allele due to the maternal imprinting. Following the initial findings, we searched for MKRN3 mutations in 213 patients with apparently sporadic CPP using polymerase chain reaction followed by direct enzymatic purification and automated sequencing (Sanger). Three new mutations and two previously reported, including four frameshifts and one missense variant was identified in six unrelated girls with CPP. All variants were not described in the two databases (1000 Genomes and Exome Variant Server). The familial segregation analysis performed in three out of these girls with apparently sporadic CPP and MKRN3 mutations confirmed the autosomal dominant inheritance with complete penetrance and exclusive transmission through the paternal allele, revealing familial inheritance in apparently sporadic cases. Most of these MKRN3 mutations were frameshifts or nonsense, leading to premature stop codons and truncated proteins, thus demonstrating positive genotype- phenotype correlation. The two missense mutations (p.Arg365Ser and p.Phe417Ile) identified were located within zinc finger motifs, regions predicted to be essential for the protein function. Besides that, all missense mutations were predicted to be pathogenic by in silico analysis. All patients carrying MKRN3 mutations exhibited typical clinical and hormonal features of premature activation of the reproductive axis. The median age of puberty onset was 6.0 years in girls (ranging from 3.0 to 6.5) and 8.0 years in boys (ranging from 5.9 to 8.5). In view of the imprinting phenomenon, methylation analysis was also performed in a subgroup of 52 patients with CPP by MSMLPA technique, but no methylation abnormalities were detected. In conclusion, our work has identified a new gene associated with CPP. Currently, inactivating mutations in MKRN3 represent the most common genetic cause of familial CPP (33%). MKRN3 is the first imprinted gene associated with pubertal disorders in humans. However, its precise mechanism of action in the regulation of GnRH secretion needs further studies

Introdução: Na adolescência ocorre importante período de desenvolvimento sexual e acentuado crescimento corporal, caracterizado por alterações na quantidade e distribuição de gordura e massa magra diferente entre os sexos com importante impacto no estado nutricional, tanto atual como futuro. Objetivo: Analisar a associação entre maturação sexual e adiposidade em crianças e adolescentes. Método: Foi realizado estudo longitudinal com 617 estudantes de 8 a 18 anos de idade de duas escolas do município de São Paulo. Foram realizadas três coletas de dados com intervalo de 6 meses.. Dados de peso, estatura, perímetro da cintura foram coletados. O excesso de peso foi classificado com base nos valores críticos do índice de massa corporal (IMC) para crianças e adolescentes brasileiros. Valores de resistência (R), reactância (Xc), ângulo de fase e composição corporal foram obtidos por meio de impedância bioelétrica. O estágio de maturação sexual (EMS) foi auto-avaliado e os indivíduos foram distribuídos em quartis de idade segundo EMS e sexo. Foram comparados os indivíduos com maturação relativamente acelerada com os demais indivíduos. Resultados: Foi observada associação negativa entre escore z do IMC e idade de início da maturação sexual em meninos. A maturação sexual relativamente acelerada se associou com excesso de peso e com maior incremento nos valores de escore z do IMC. Meninas com maturação relativamente acelerada apresentaram maior adiposidade central. A maturação acelerada se associou a R/E, Xc/E e ângulo de fase e quantidade absoluta de massa magra. Menores vetores de impedância foram observados em meninas com maturação acelerada. Conclusão: A maturação sexual acelerada associouse a alterações nos parâmetros bioelétricos, composição corporal, maior incremento no IMC e obesidade em crianças e adolescentes
Adolescence is considered an important period of sexual development and an increase body-growth, characterized by changes in the amount and distribution of fat and lean mass distinct between sexes, with a significant impact on nutritional status, current and future. Objective: To evaluate the association between sexual maturation and adiposity in children and adolescents. Method: We conducted a longitudinal study with 617 students 8-18 years old from two schools in São Paulo. Were performed three data collections at intervals of six months. Data on weight, height, waist circumference were collected. Overweight was classified based on cutoffs of body mass index (BMI) for Brazilian children and adolescents. Resistance (R), reactance (Xc), phase angle (PA) and body composition values were obtained by bioelectrical impedance. The sexual maturation stage (SMS) was self-assessed and subjects were divided into quartiles of age second sex and SMS. We compared the subjects with relatively rapid maturation with other individuals. Results: We observed a negative association between BMI z score and age of onset of sexual maturation in boys. The relatively rapid sexual maturation was associated with overweight and greater increase in the values of z scores of BMI. Girls with relatively rapid maturation had higher central adiposity. The accelerated maturation was associated with R/H, Xc/H and PA and the absolute amount of lean mass. Lower impedance vectors were observed in girls with accelerated maturation. Conclusion: The accelerated sexual maturation was associated with changes in bioelectrical parameters, body composition, greater increase in BMI and obesity in children and adolescents

A kisspeptina, codificada pelo gene KISS1, é um neuropeptídeo crucial na regulação do início da puberdade. A kisspeptina estimula a secreção hipotalâmica do hormônio liberador de gonadotrofinas (GnRH) após se ligar ao seu receptor GPR54. Mutações inativadoras do GPR54 são atualmente consideradas como uma causa rara de hipogonadismo hipogonadotrófico isolado (HHI) normósmico. Recentemente, uma mutação ativadora no receptor GPR54 foi implicada na patogênese da puberdade precoce dependente de gonadotrofinas (PPDG). Com base nesses achados, levantamos a hipótese de que alterações no gene KISS1 poderiam contribuir para a patogênese de distúrbios puberais centrais. O objetivo do presente estudo foi investigar a presença de variantes no gene KISS1 em pacientes com PPDG e HHI. Sessenta e sete crianças brasileiras com PPDG (63 meninas e 4 meninos) e 61 pacientes com HHI (40 homens e 21 mulheres) foram selecionados, incluindo casos esporádicos e familiares em ambos os grupos. A população controle consistiu de 200 indivíduos com história de desenvolvimento puberal normal. A região promotora e os 3 exons do gene KISS1 foram amplificados e submetidos a sequenciamento automático. Duas novas variantes no gene KISS1, p.P74S e p.H90D, foram identificadas em duas crianças não relacionadas, portadoras de PPDG idiopática. Ambas as variantes estão localizadas na região amino-terminal da kisspeptina-54 e estavam ausentes em 400 alelos controles. A variante p.P74S foi identificada em heterozigose em um menino que desenvolveu puberdade com um ano de idade. Sua mãe e avó materna, que apresentavam história de desenvolvimento puberal normal, eram portadoras da mesma variante em heterozigose, sugerindo penetrância incompleta e/ou herança sexo-dependente. A variante p.H90D foi identificada em homozigose em uma menina com PPDG, que desenvolveu puberdade aos seis anos de idade. Sua mãe, com história de menarca aos dez anos de idade, era portadora da mesma variante em heterozigose. Células transfectadas estavelmente com GPR54 foram estimuladas com concentrações crescentes de kisspeptina-54 (kp-54) humana selvagem ou contendo as mutações (kp-54 H90D e kp-54 P74S) e o acúmulo de fosfato de inositol (IP) foi medido. Nos estudos in vitro, a kp-54 P74S apresentou uma capacidade de ativação do receptor GPR54 semelhante à kp-54 selvagem. A kp-54 p.H90D mostrou uma ativação da sinalização do receptor significativamente mais potente que a kp-54 selvagem, sugerindo que essa é uma mutação ativadora. No grupo de HHI, uma nova variante (c.588-589insT) foi identificada em heterozigose na região 3 não traduzida do gene KISS1 em um paciente do sexo masculino. O papel dessa variante no fenótipo de HHI permanece indeterminado. Em conclusão, duas mutações no gene KiSS1 foram descritas pela primeira vez em associação com PPDG.
Kisspeptin, encoded by the KISS1 gene, is an important regulator of puberty onset. After binding to its receptor GPR54, kisspeptin stimulates gonadotropin-releasing hormone secretion by the hypothalamic neurons. Inactivating GPR54 mutations are a rare cause of normosmic isolated hypogonadotropic hypogonadism (IHH). Recently, a unique GPR54 activating mutation was implicated in the pathogenesis of gonadotropin dependent precocious puberty (GDPP). Based on these observations, we hypothesized that mutations in the KISS1 gene might be associated with central pubertal disorders. The aim of this study was to investigate KISS1 mutations in idiopathic GDPP and normosmic IHH. Sixty-seven Brazilian children (63 girls and 4 boys) with idiopathic GDPP and 61 patients with normosmic IHH (40 men and 21 women) were selected. Familial and sporadic cases were included in both groups. The control population consisted of 200 individuals who had normal timing of puberty. The promoter region and the 3 exons of the KISS1 gene were amplified and automatically sequenced. Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in two unrelated children with idiopathic GDPP. Both mutations were absent in 400 control alleles and are located in the amino-terminal region of kisspeptin-54. The p.P74S mutation was identified in the heterozygous state in a boy who developed puberty at 1 yr of age. His mother and maternal grandmother, who had normal pubertal development, were also heterozygous for the p.P74S mutation, suggesting incomplete penetrance and/or sex-dependent inheritance. The p.H90D mutation was identified in the homozygous state in a girl with GDPP, who developed puberty at 6 yr of age. Her mother, who had menarche at 10 yr of age, carried the p.H90D mutation in the heterozygous state. CHO cells stably transfected with GPR54 were stimulated with different concentrations of synthetic human wild type or mutant kisspeptin-54 (KP54) and inositol phosphate (IP) accumulation was measured. In vitro studies revealed that the capacity of the p.P74S mutant KP54 to stimulate IP production was similar to the wild type. The p.H90D kisspeptin-54 showed a significantly more potent activation of GPR54 signaling in comparison to the wild type in vitro, suggesting a gain-of-function mutation. In the IHH group, a heterozygous variant in the 3 UTR of the KISS1 gene (c.588-589insT) was identified. The role of this variant in the IHH phenotype remains to be determined. In conclusion, two KiSS1 mutations were described for the first time in association with GDPP.

The aim of the thesis was to identify whether children with a diagnosis of Premature Adrenarche (PA) or Central Precocious Puberty (CPP) presented with an atypical psychological profile in comparison to typically-developing children. A battery of psychometrics was constructed to study several domains, including eating behaviour, self-perception and intellectual ability. Measures of family environment and parental stress were also included. In addition, an interpretative phenomenological analysis was conducted on five interviews with parents to gain a greater insight into the experience of parenting a child with a diagnosis of early puberty. It was found that several differences between groups, such as weight gain, internalising behaviours and sleep problems, could be attributed to hormonal or behavioural changes typically associated with pubertal development across all groups. Other observations were specific to the pubertal disorders, such as risk of obesity, problem eating behaviours, anxiety and depression, and aggression. Furthermore, being from a family with a single-parent or non-parent care-giver, and increased family stress were related to earlier pubertal development. In summary, children with a diagnosis of PA or CPP may be more likely to display altered behaviour and psychopathology, but some of these difficulties may also occur in typical pubertal development.

Objetivou-se no presente estudo, avaliar o efeito da nutrição proteica no terço final da gestação de vacas Nelore, seguido de estratégias de suplementação e/ou alimentação de suas crias fêmeas sobre a idade à puberdade até os 18 meses. O delineamento utilizado foi o inteiramente casualizado em esquema fatorial 2x2x2; constituindo-se de: 2 manejos suplementar das vacas no pré-parto (Fase I); 2 manejos suplementar das crias (Fase II) e 2 manejos alimentar na recria (Fase IIIA). Na Fase I, 241 vacas foram suplementadas com farelos de soja na proporção de 0,5kg/vaca/dia (Tratamento 1) e 258 vacas foram mantidas sem acesso a suplementação Tratamento 2 (controle - Fase I). Cerca de metade do número de vacas e suas crias fêmeas, nascidas na Fase I, foram distribuídas em dois tratamentos na Fase II-(suplementação das crias) aos 110 dias de idade das crias, as quais passaram receber ou não uma mistura mineral proteica energética em creep-feeding, constituindo assim, os tratamentos suplementação de bezerras em creep-feeding (n =119) e controle (sem suplementação, n =122 ) na Fase II até os 205 dias de idade (desmama). Na fase III A, metade das bezerras de cada grupo na fase II foram manejados em confinamentos (n=119) e a outra metade permaneceu no pasto (Grupo controle, n = 122), até os 320 dias de idade. Na fase IIIB, todas as novilhas foram manejados juntos a pasto e submetidas a estação de monta a partir dos 440 dias aos 560 dias de idade. Os sistemas de suplementação das vacas e bezerras na fase de cria não afetaram o peso corporal, concentração de IGF-1 e percentual de peso adulto das novilhas no início da estação de monta (P > 0,05). O manejo das novilhas em confinamento na fase IIIA, aumentou o número de novilhas púberes (31,9% vs 13,9%; P < 0,01), para as novilhas alimentadas ou não em confinamento, respectivamente. Entretanto, a alimentação em confinamento não ocasionou diferença na idade que estas atingiram a puberdade. Considerando apenas as novilhas que atingiram à puberdade (n = 55) houve efeito de interação entre as fases de suplementação/alimentação e a idade à puberdade (P < 0,05). Para as vacas manejadas na Fase I, a suplementação influenciou a ciclicidade das mesmas no momento da IATF (68,9% vs 55,4%; P < 0,05), porém não ocasionou diferença no número de vacas prenhes (60,1 vs 55,3%; P > 0,05), vacas suplementadas e não suplementadas, respectivamente. Similarmente, a suplementação das novilhas em creep-feeding, não influenciou a taxa de prenhez das vacas (P > 0,05). Em conclusão, a suplementação das vacas com fontes proteicas não influenciou a idade a puberdade de novilhas até os 18 meses, sendo que o manejo alimentar em confinamento aumentou o número de novilhas púberes em relação com as manejadas no pasto.
The aim of the present study was to evaluate the effect of protein nutrition in the final third of gestation of Nelore cows, followed by supplementation strategies and / or feed their young females over the age of puberty until 18 months. The experimental design was a completely randomized factorial 2x2x2; constituting of: 2 suplementary managements of cows in pre-partum (Phase I); 2 supplementary managements of offspring (Phase II) and 2 alimentary managements during rearing (Phase IIIA). In Phase I, 241 cows were supplemented with soybean meal at the rate of 0.5 kg / cow / day (Treatment 1) and 258 cows were kept without access to supplemental Treatment 2 (control - phase I). About half of the cows and their female offspring, born in Phase I, were assigned to two treatments in Phase-II (supplementation of cubs) at 110 days of age of the offsprings, which now receive or not a mineral mix protein energy in creep-feeding, constituting, treatments supplementation in calves creep-feeding (n = 119) and control group (without supplementation, n = 122) in Phase II until 205 days of age (weaning). In phase III A, half of each group of calves in phase II were managed in feedlots (n = 119) and the other half remained in the pasture (control group, n = 122), up to 320 days of age. In stage IIIB, all heifers were managed together and submitted to the pasture breeding season from 440 days to 560 days old. Supplementation systems of cows and calves during the growing period did not affect body weight, concentration of IGF-1 and percentage of mature weight of heifers at the beginning of the breeding season (P> 0.05). The management of heifers in the feedlot phase IIIA, increased the number of pubertal heifers (31.9% vs 13.9%, P <0.01) for heifers fed in confinement or not, respectively. However, feeding in confinement caused no difference in age they reached puberty. Considering only heifers reached puberty (n = 55) there was an interaction effect between phases of supplementation / nutrition and age at puberty (P <0.05). For cows managed in Phase I, supplementation influenced the cyclicality of the same at the time of TAI (68.9% vs 55.4%, P <0.05), but caused no difference in the number of pregnant cows (60.1 vs 55.3%, P> 0.05), supplemented and nonsupplemented cows, respectively. Similarly, the addition of heifers in creep-feeding did not influence the rate of pregnancy in cows (P> 0.05). In conclusion, supplementation of cows with protein sources did not influence the age at puberty in heifers up to 18 months, and feeding management in confinement increased the number of pubertal compared with those managed on pasture.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Puberdade precoce central em meninas à definida como desenvolvimento puberal causado pela ativaÃÃo do eixo hipotÃlamo-hipÃfise-gonadal antes de 8 anos de idade. Nestas condiÃÃes, a estimulaÃÃo das gonadotrofinas produz aumento dos ovÃrios e a secreÃÃo de estrogÃnio resulta em aumento uterino. O exame de ultra-som pÃlvico prova ser um mÃtodo preciso e nÃo invasivo na investigaÃÃo da genitÃlia interna de pacientes do sexo feminino. Ultra-sonografia pÃlvica foi sistematicamente realizada em 18 meninas com diagnÃstico de puberdade precoce central idiopÃtica para avaliar o impacto do tratamento com anÃlogo de GnRH na genitÃlia interna feminina. Antes e, em mÃdia, 3 meses apÃs o inÃcio do tratamento, foram avaliados os volumes uterino e ovarianos, o diÃmetro longitudinal do Ãtero, eco endometrial e grau de maturidade de Tanner. Os nossos dados demonstraram que o Ãtero e os ovÃrios estÃo aumentados na Ãpoca do diagnÃstico. ApÃs, aproximadamente 3 meses de terapia, ambos os volumes, uterino e ovarianos, reduziram seus valores, o comprimento do Ãtero diminuiu e houve uma regressÃo quanto ao estÃgio puberal de Tunner. Quanto ao eco endometrial nÃo houve mudanÃa significativa. Os nossos resultados confirmam a ecografia pÃlvica como uma ferramenta confiÃvel para investigaÃÃo da genitÃlia interna em meninas com puberdade precoce e como valioso mÃtodo para avaliaÃÃo da eficÃcia do tratamento com anÃlogo de GnRH.
Central precociuos puberty in girls is defined as pubertal development caused by activation of the hypothalamic-pituitary-gonadal axis before 8 years old. In this condition, gonadotropin stimulation produces ovarian enlargement and estrogen secretion results in uterine enlargement. Pelvic ultrasound has proven to be an accurate and noninvasive technique for investigation of internal genitalia in female patients. Pelvic ultrasonography was systematically perfomed on 18 girls with idiopathic central precociuos puberty to investigate the impact of treatment with gonadotropin-releasing hormone analogues on female internal genitalia. Before and after three months of treatment were evaluated ovarian and uterine volumes, uterine lenght, endometrial stripe and Tanner staging. Our data demonstrated that ovaries and uterus are enlarged at the time of diagnosis. Later, average 3 months of treatment, both ovarian and uterine volumes decreased, the uterine lenght decreased and the Tanner staging regressed. The endometrial echogenicity did not showed changes. Ours results confirmed pelvic ultrasonography as a reliable tool for investigation of internal genitalia in girls with precociuos puberty and as a valid method for evaluation of the efficacy of treatment with gonadotropin-releasing hormone analogues.

碩士
國立中興大學
資訊管理學系所
105
Puberty is a complex, dynamic and developmental transition from the juvenile to adult feature on somatic cells and sexual maturity, which also influenced by multiple genes and internal secretion. Precocious Puberty is defined as secondary sexual characteristics of children appear before eight to nine years old, which also known as puberty develop in advance. There are many types of Precocious Puberty (1: Central Precocious Puberty, 2: Peripheral Precocious Puberty), the study focus on Central Precocious Puberty. In recent years, the incidence rate of Precocious Puberty is increasing due to improvement of life. In Taiwan, most of the researches of Precocious Puberty focus on the reason of the disease, but no follow up in cure. 　　The study analyze 182 patients from the Division of Pediatrics in a hospital of central Taiwan between Oct 1992 and May 2004. The study propose a hybrid-classification-based model of CPP identification, which include decision tree (DT), Naive Bayes Classifiers (NB), Classification and Regression Tree (CART), Random Tree (RT) and Multi-Layer Perception Neural Network (MLP). The result show that the mother part may affect the disease, the prediction models constructed by C4.5, MLP also have good performance on bone age and the area under the ROC curve (AUC) are up to 0.78. After validated by a greater amount of data, our study could be valuable in clinical practice.

BACKGROUND: MKRN3 is a gene recently identified to encode the first known inhibitor of puberty initiation. MKRN3 mutations have been identified in children diagnosed with familial central precocious puberty. MKRN3 is a maternally imprinted gene; only the father’s allele is expressed by the child. Family studies and patterns of inheritance affirm that mutant alleles only result in CPP when the mutation was inherited from the father. Although mutations in MKRN3 were found to have implications in development of central precocious puberty, its mechanism of action remains a mystery. Previous studies in our laboratory have focused on MKRN3’s function as an E3 ubiquitin ligase and thus sought to investigate its potential targets in the cell. Because kisspeptin is the most potent known activator of GnRH neurons, it seemed to be the most likely candidate. Despite efforts to identify an association between kisspeptin levels and MKRN3 expression, little headway has been made. OBJECTIVE: Beyond the most common known function of E3 ubiquitin ligases in protein degradation, increasing evidence suggests that E3 ubiquitin ligases contribute to aiding human brain development throughout childhood and into adolescence. This proposed action of E3 ubiquitin ligases led us to propose a role for MKRN3 as a potential regulator of GnRH neuronal plasticity and maturation. I hypothesized that MKRN3 plays a role in delaying GnRH neuronal maturation and neuronal plasticity until puberty onset. Thus, MKRN3 deficiency would result in premature GnRH neuron maturation. METHODS: A mouse model of Mkrn3 knockout mice (Mkrn3 +/p-) was compared to wildtype mice (Mkrn3+/+). Using DAB IHC, GnRH neurons in the rostral pre-optic area (rPOA) area were labeled and morphologically analyzed. Additionally, Golgi staining of neurons in the arcuate nucleus was done to visualize details of neuronal synapses. Neurons were visualized under 40X and 100X magnification. To visualize spines, confocal images of dendrites from Mkrn3+/p- and Mkrn3+/+ mice were captured. Images were visualized and analyzed using both ImageJ and NeuronStudio software (for spine analysis only). RESULTS: Initial morphological analysis of GnRH neurons failed to show any significant effect of the Mkrn3 genotype on dendrite arrangement, varying among complex, bipolar, and unipolar. Similarly, Golgi staining analysis revealed that the Mkrn3 genotype did not have a measurable effect on dendritic spine type percentages. Mkrn3+/p- and Mkrn3+/+ mice showed no apparent difference in the percentage of stubby, thin, and mushroom shaped spines. However, Mkrn3 deficiency did show an effect on spine density, with neurons from the arcuate nucleus of the hypothalamus of Mkrn3+/p- mice having an increased total number of dendritic spines compared to neurons from wildtype mice. CONCLUSIONS: Mkrn3 does not appear to have an effect on dendrite architecture of GnRH neurons nor change in spine type of arcuate nucleus neurons. However, the Mkrn3 genotype does seem to affect neuronal spine density, at least in the population of neurons in the arcuate nucleus. Future research is needed to conclusively determine the mechanism of action of MKRN3 and its specific role in central precocious puberty.

碩士
國立成功大學
環境醫學研究所
97
This research is aimed to investigate the relationships between urinary monoester metabolites and the PAE levels in house dust for precocious puberty girls and normal girls. The demographic characteristics and exposure scenario including dietary ingestion, personal care products and cosmetic usage for subjects were obtained from an interviewed questionnaire. The precocious puberty girls were recruited from the policlinic of Pediatric Endocrinology in National Cheng Kung University Hospital and normal girls were recruited as control group. The urinary monoester metabolites of subjects and the phthalate ester levels in house dust were measured by HPLC-MS/MS and GC/MS, respectively. The analytic results from all house dust samples in four different areas shows that DEHP (834.7~1643.6 mg/kg) is the dominant compound in all samples, followed by DBP (21.5~34.4 mg/kg) and BBP(3.03~4.16 mg/kg). DEHP contributes over 96% of total PAE concentration in house dust samples. The levels of DBP in the dust from bed of precocious puberty girls were significantly higher than those from normal girls (P=0.02). In total, the levels of DEHP in house dust in Taiwan were 2.3-4.8 folds higher than those in German, US and Norway. The highest PAE metabolites in urine samples from all subjects were MBP (78.1 µg/g creatinine), followed by MEP (18.7 µg/g creatinine), MEHP (12.9 µg/g creatinine), MMP (9.09 µg/g creatinine) and MBzP (5.25 µg/g creatinine). The levels of MEP in precocious puberty girls were significantly higher than those in normal girls(25.5 vs.10.9 µg/g creatinine，P=0.013), while the MBP was on the contrary. In general, the internal dose of MBP in this study groups were 2 times higher than American children and the internal dose of MEHP were 2.5 times higher than American children and German childen, respectively. In the dietary comsumption survey, the consumption quantities of beef, mutton, milk products, and hot dog in precocious puberty girls were higher than normal girls. The plastic packaged convenient foods were consumed more frequently in precocious puberty girls than normal girls. All subjects were divided into high and low PAE level groups by median level of house dust and adjusted-urinary samples. The data shows that the high PAEs in house dust group used more plastics materials as flooring material than low level group. The consumption quantities of off-premises eating, off-premises drinking and the usage of personal care products in high urinary PAE group were higher than low PAE group. Futhermore, there was no relationships between urinary monoester metabolite concentrations and dust PAE levels in precocious puberty girls. The daily intake dose (DIest) of five PAEs in all subjects were estimated from the level of urinary PAE metabolites based on the pharmacokinetics model. The calculated median DIest (µg/kg/day) of five PAEs in all girls were as followed: DEHP was 5.21(0.38-74.36) µg/kg/day, DBP was 2.69(0.77-15.54) µg/kg/day, DEP was 0.64(0.04-13.05) µg/kg/day, DMP was 0.31(0.03-4.42) µg/kg/day and BBP was 0.17(0.03-2.75) µg/kg/day, respectively.

碩士
國立成功大學
環境醫學研究所
98
In recent years, studies have shown the incidence of precocious puberty increased year by year, especially for girls. The epidemiological studies found that phthalate esters (PAEs) exposure may be related to the onset of puberty. PAEs were widely used in PVC plastic products, food containers, personal hygiene and commercial products. The significant estrogenic activities of various PAEs have been observed in different studies. While the PAEs entered the body, it acted as an estrogen analog and bound to estrogen receptor α (ERα). In addiction, several evidences have shown that Kisspeptins were the peptide products of the Kiss1 gene, and its receptor GPR54 (G protein coupled receptor) plays a crucial role in governing reproductive endocrine system and the onset of puberty. Moreover, the ERα regulated the release of GnRH (Gonadotropin -releasing hormone) by interacted with estrogen and therefore played an important role in the feedback loop of Kisspeptin/GPR54 system. In this study, we investigate the association among phthalate exposure, kisspeptin 54 peptide level and Kiss-1/GPR54 gene polymorphism on early onset of girl puberty. The objectives were: (1) to evaluate exposure factor of PAEs in daily life by standardized questionnaires and to assess the internal exposure dose by analyzing of PAEs metabolites in urine; (2) to assess the differences of Kisspeptin54 peptide between two groups; (3) to study the relationship of Kisspeptin54 peptide secretion and degree of sex characteristic development; (4) to assess the relationship of PAEs metabolite concentrations and Kisspeptin54 peptide; (5) to explore the Kiss-1, GPR54 gene sequence variation between case and control, and to assess the protein sequence variation using relevant biological information database. We recruited the precocious puberty girls from the National Cheng Kung University Hospital pediatric clinic as case group, and the control group recruited by voluntary participation of healthy girls. The total of 40 precocious puberty girls and 11 normal girls were recruited in this study. Seven urinary metabolites of PAEs were analysed by HPLC-MS/MS, and the commercial radio immunoassay kit was used for Kisspeptin54 analysis. We found that the higher frequencies and quantities of eating greasy meat, fried foods, beverages, snacks and nutriment, off-premises eating and the usage of PVC plastic wrap were the potential causes of higher phthalate exposure. Futhermore, living in the aged building and less house cleaning may also contribute to higher indoor PAEs exposure. The concentration of seven metabolites of PAEs in urine from precocious puberty girls were higher than normal girls, and the MMP, MBP, MBzP, MEHHP and MEOHP showed significant difference between two groups(MMP:8.35 vs. 4.31 ?g/L, P=0.013; MBP: 115.10 vs. 32.12 μg/L , P = 0.0002; MEHHP: 64.86 vs. 27.53 μg/L, P = 0.003; MEOHP: 59.98 vs. 25.73 μg/L, P = 0.003; MBzP: 11.17 vs. 2.79 μg/L, P = 0.0001). After divided all subjects into three development category groups, the MMP, MBP, MBzP, MEHHP and MEOHP concentration were also increased significantly. While adjusted with creatinine, only the MBP and MBzP have show the increased trend (P ＜0.05). We also calculated the aggregated dose to estimate the internal estrogenic activities of PAEs due to the different ERα binding affinities of PAEs. We found that the aggregated dose of precocious puberty girls (6.47 (2.70-16.75) μg/kg/day) was significantly higher than those of normal girls (2.99 (1.04-10.28) μg/kg/day, P=0.0007). It showed that the higher phthalate exposure in precocious puberty girls may cause more internal estrogenic activites than normal girls. The average Kisspeptin54 concentrations were 2.15 (1.39-2.86) and 1.95 (1.69-2.18) pmol/L (P=0.06) for precocious puberty girls and normal girls, respectively. Moreover, the Kisspeptin54 concentrations were also significantly increased with sexual development. We also found that Kisspeptin54 secretion was significantly correlated with the peak lutenising hormone (LH) concentration analyzed by LHRH test (r=0.4, P= 0.03). The significant correlation between Kisspeptin54 secretion and urinary MMP, MBP and MBzP concentrations (uncorrected for creatinine) were also observed, however, only the correlation between Kisspeptin54 and urinary MBP remained significant after creatinine correction (r=0.537, P =0.0006). The results of gene sequencing showed that 16 and 14 sequence variants in Kiss-1 and GPR54 gene, respectively, were identified in this study. Furthermore, 9 of 16 and 6 of 14 sequence variants in Kiss-1 and GPR54 gene, respectively, were confirmed SNP. Three sequence variants among them could lead to amino acid change in both two genes. For allele and genotype frequency of sequence variants comparison, no significant differences were found between case and control groups. In conclusion, the difference of expression of two genes could be the important index of gene variance. Further studies and more samples are needed to clarify the relationship between precocious puberty and the polymorphism of these genes.

Introdução: Os agonistas da hormona libertadora de gonadotrofinas (GnRHa) são usados no tratamento da puberdade precoce central (PPC). Muitos estudos têm sido realizados para avaliar o impacto dos GnRHa no índice de massa corporal (IMC) dessas crianças, mas os resultados são inconsistentes. O objetivo deste estudo é avaliar o efeito do tratamento com GnRHa sobre o IMC de raparigas com diagnóstico de PPC. Métodos: Foi realizado um estudo longitudinal retrospetivo com trinta e sete raparigas, diagnosticadas com PPC. Estas foram divididas em dois grupos, um grupo de meninas tratadas com GnRHa e um grupo controlo que não foi submetido a tratamento. Considerando o seu IMC inicial, as raparigas tratadas foram também divididas num grupo com peso normal (PN) e outro grupo com excesso de peso (EP)/obesidade. Resultados: No final do tratamento, não se verificou uma diferença estatisticamente significativa entre o IMC z-score do grupo tratado e o IMC z-score do grupo não tratado (p>0.05). Dentro do grupo tratado, a variação do IMC z-score entre o início e o final do tratamento não foi estatisticamente significativa (p>0.05), assim como no grupo não tratado (p>0.05). Durante este período, não se registaram alterações estatisticamente significativas no peso z-score e no IMC z-score, tanto no grupo com PN tratado como no grupo com EP/obesidade tratado (p>0.05). Conclusões: O tratamento da PPC com GnRHa não condicionou um aumento do IMC z-score dos pacientes. Concluímos também que o IMC z-score inicial dos pacientes não influencia a variação do IMC z-score ao longo do tratamento.
Background: Gonadotropin-releasing hormone agonists (GnRHa) are used to treat central precocious puberty (CPP). Many studies were performed to assess the impact of GnRHa on the body mass index (BMI) of these children, but the results are inconsistent. The aim of this study is to evaluate the effect of GnRHa treatment on the BMI of girls diagnosed with CPP. Methods: A retrospective longitudinal study was carried out with thirty-seven girls diagnosed with CPP. These were divided into two groups, a group of girls treated with GnRHa and a control group that did not undergo treatment. Considering their initial BMI, treated girls were also divided into normal weight (NW) group and overweight (OW)/obesity group. Results: At the end of treatment, there was no statistically significant difference between the BMI z-score of the treated group and the BMI z-score of the untreated group (p>0.05). Within the treated group, BMI z-score variation between the beginning and the end of the treatment was not statistically significant (p>0.05), as well as in the untreated group (p>0.05). During this period, there were no statistically significant changes in the weight z-score and the BMI z-score, both in the treated NW group and in the treated OW/obesity group (p>0.05). Conclusions: The GnRHa treatment of CPP did not condition an increase in girls' BMI z-score. We also concluded that the initial BMI z-score of the patients does not influence the variation of the BMI z-score throughout the treatment.

Introdução: Os agonistas da hormona libertadora de gonadotrofinas (GnRHa) são usados no tratamento da puberdade precoce central (PPC). Muitos estudos têm sido realizados para avaliar o impacto dos GnRHa no índice de massa corporal (IMC) dessas crianças, mas os resultados são inconsistentes. O objetivo deste estudo é avaliar o efeito do tratamento com GnRHa sobre o IMC de raparigas com diagnóstico de PPC. Métodos: Foi realizado um estudo longitudinal retrospetivo com trinta e sete raparigas, diagnosticadas com PPC. Estas foram divididas em dois grupos, um grupo de meninas tratadas com GnRHa e um grupo controlo que não foi submetido a tratamento. Considerando o seu IMC inicial, as raparigas tratadas foram também divididas num grupo com peso normal (PN) e outro grupo com excesso de peso (EP)/obesidade. Resultados: No final do tratamento, não se verificou uma diferença estatisticamente significativa entre o IMC z-score do grupo tratado e o IMC z-score do grupo não tratado (p>0.05). Dentro do grupo tratado, a variação do IMC z-score entre o início e o final do tratamento não foi estatisticamente significativa (p>0.05), assim como no grupo não tratado (p>0.05). Durante este período, não se registaram alterações estatisticamente significativas no peso z-score e no IMC z-score, tanto no grupo com PN tratado como no grupo com EP/obesidade tratado (p>0.05). Conclusões: O tratamento da PPC com GnRHa não condicionou um aumento do IMC z-score dos pacientes. Concluímos também que o IMC z-score inicial dos pacientes não influencia a variação do IMC z-score ao longo do tratamento.
Background: Gonadotropin-releasing hormone agonists (GnRHa) are used to treat central precocious puberty (CPP). Many studies were performed to assess the impact of GnRHa on the body mass index (BMI) of these children, but the results are inconsistent. The aim of this study is to evaluate the effect of GnRHa treatment on the BMI of girls diagnosed with CPP. Methods: A retrospective longitudinal study was carried out with thirty-seven girls diagnosed with CPP. These were divided into two groups, a group of girls treated with GnRHa and a control group that did not undergo treatment. Considering their initial BMI, treated girls were also divided into normal weight (NW) group and overweight (OW)/obesity group. Results: At the end of treatment, there was no statistically significant difference between the BMI z-score of the treated group and the BMI z-score of the untreated group (p>0.05). Within the treated group, BMI z-score variation between the beginning and the end of the treatment was not statistically significant (p>0.05), as well as in the untreated group (p>0.05). During this period, there were no statistically significant changes in the weight z-score and the BMI z-score, both in the treated NW group and in the treated OW/obesity group (p>0.05). Conclusions: The GnRHa treatment of CPP did not condition an increase in girls' BMI z-score. We also concluded that the initial BMI z-score of the patients does not influence the variation of the BMI z-score throughout the treatment.

Humans experience ubiquitous exposures to estrogenic environmental chemicals from food, personal care products, and other industrial and consumer goods. Bisphenol A (BPA), a well-studied xenoestrogen, is known to alter development of estrogen-sensitive organs including the brain, reproductive tract, and mammary gland. Bisphenol S (BPS), which has a similar chemical structure to BPA, is also used in many consumer products, but its effects on estrogen-sensitive organs in mammals has not been thoroughly examined. In our study, pregnant CD-1 mice were orally exposed to BPS or ethinyl estradiol (EE2, a positive control for estrogenicity) from gestational day 9 through postnatal day (PND) 2, the period when many estrogen-sensitive organs are developing. After weaning, the offspring were administered either oil (vehicle) or an estrogen challenge (1 μg EE2/kg/day) for ten days starting at PND21 (prior to puberty), PND80 (early adulthood), or PND260 (later adulthood). Timing of puberty was evaluated in females by noting the date on which vaginal opening occurred. After the 10 day estrogen challenge, we evaluated the response of endocrine sensitive organs through measurements of organ weight, tissue morphology, and gene expression in both males and females. We observed dose- and sex-specific effects of BPS and EE2 treatment, as well as alterations in the responses of males and females to the estrogen challenge. This study sheds light on the effects of low dose xenoestrogen exposures on estrogen-sensitive organs including the reproductive tract and mammary gland. Furthermore, it improves our understanding of the influence of environmental chemicals on secular trends of earlier age of puberty in girls reported over the past few decades.