In mammals, the most frequently observed hybrid dysgenesis effects are growth disturbances and male sterility. Profound defects in placental development have been described and our work on hybrids in genus Mus has demonstrated putative hybrid dysgenesis effects that lead to defects in lipid homeostasis and maternal behavior. Interestingly, mammalian interspecies hybrids exhibit strong parent-of-origin effects in that offspring of reciprocal matings, even though genetically identical, frequently exhibit reciprocal phenotypes. Recent studies have provided strong link between epigenetic regulation and growth, behavior and placental development. Widespread disruption of genomic imprinting has been described in hybrids between closely related species of the genus Peromyscus. The studies presented in this thesis aim to investigate the effects of disrupted epigenetics states on altered growth, female infanticide and placental dysplasia observed in Mus hybrids. We showed that loss-of-imprinting (LOI) of a paternally expressed gene, Peg1, was correlated with increased body weight of F1 hybrids. Furthermore, we investigated whether LOI of Peg1 in F1 females would interfere with maternal behavior. A subset of F1 females indeed exhibited highly abnormal maternal behavior in that they rapidly attacked and killed the pups. By microarray hybridization, a large number of differentially expressed genes in the infanticidal females as compared to normally behaving females were identified. In addtion to Peg1 LOI, we studied allelic expression of numerous imprinted genes in adult Mus interspecies hybrids. In contrast to the study from Peromyscus, patterns of LOI were not consistent with a direct influence of altered expression levels of imprinted genes on growth. Finally, we investigated the allelic interaction between an X-linked locus and a paternally expressed gene, Peg3, in placental defects in Mus hybrids. This study further strengthened the notion that divergent genetic and epigenetic mechanisms may be involved in hybrid dysgenesis in diverse groups of mammals.

Different species are separated by pre-zygotic reproductive barriers which impede gene flow between them. Rarely, when pre-zygotic barriers break down, interspecific hybrids are produced that display abnormal phenotypes, collectively called hybrid dysgenesis effects. Interspecies hybrid placental dysplasia (IHPD) in the genus Mus is a very consistent X-linked hybrid dysgenesis effect. Reproductive cloning and mutation of the gene Esx1 lead to placental hyperplasias with phenotypic similarities to IHPD. Comparative gene expression analysis of these three different models of placental hyperplasia showed that different mechanisms underlie these placental hyperplasias. We also identified several genes for which roles in placentation had not been studied earlier. We screened five of these genes, Car2, Ncam1, Fbln1, Cacnb3 and Cpe for their functions in placentation. Analysis of the spatio-temporal expression patterns of these genes during mouse placental development showed that they are ectopically expressed in IHPD placentas. Placental phenotype and gene expression was then studied in mice mutant for these genes. Our results show that complicated by the expression of functional counterparts, deletion of these genes failed to produce any consistent phenotype. Incompletely penetrant phenotypes were found in Cacnb3 and Cpe mutants. The Cpe mutant placentas recapitulated some IHPD phenotypes, despite co-expression of Cpd, a functionally redundant gene. Deregulated expression of Cpe and Cpd prior to manifestation of IHPD phenotype indicated that these are causally involved in IHPD and might be speciation genes in the genus Mus. We found that AT24 placentas also exhibit deregulated expression of these genes and could be used as a model to study IHPD. We tried rescuing the AT24 placental phenotype, by decreasing the expression of the over expressed genes. Normalization of transcript levels of these genes did not rescue the AT24 phenotype, thus indicating that up-regulation of these genes is a down-stream event in the generation of IHPD.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-07-13T11:47:34Z No. of bitstreams: 1 Taíse Lima de Oliveira Cerqueira Estudo... 2016.pdf: 4519247 bytes, checksum: 571b6a53cf6e9f3fb58c96cdf62cacb8 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-07-13T11:55:05Z (GMT) No. of bitstreams: 1 Taíse Lima de Oliveira Cerqueira Estudo... 2016.pdf: 4519247 bytes, checksum: 571b6a53cf6e9f3fb58c96cdf62cacb8 (MD5) / Made available in DSpace on 2016-07-13T11:55:05Z (GMT). No. of bitstreams: 1 Taíse Lima de Oliveira Cerqueira Estudo... 2016.pdf: 4519247 bytes, checksum: 571b6a53cf6e9f3fb58c96cdf62cacb8 (MD5) Previous issue date: 2016 / CNPq / FAPESB / Fundação Gonçalo Moniz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / INTRODUÇÃO: Hipotireoidismo Congênito (HC), é uma das doenças metabólicas mais comuns na infância com incidência de 1:3.000 a 1:4.000 recém-nascidos. Um grupo de doenças relacionadas às alterações no desenvolvimento da tireoide, denominadas disgenesias tireoidianas (DT), responsabiliza-se por aproximadamente 85% de todos os casos de HC, sendo sua patogênese pouco conhecida. OBJETIVOS: Geral: Caracterização clínica e genética de pacientes com HC diagnosticados com disgenesia tireoidiana. Específicos: 1. Caracterizar clínica dos indivíduos com HC em acompanhamento na APAE/Salvador (Associação de Pais e Amigos dos Excepcionais); 2. Avaliar a existência de associação entre malformações tireoidianas e malformações cardiacos; 3. Pesquisar polimorfismos e mutações nos genes candidatos: PAX8, TSH-R, NKX2.5 e HES1, em pacientes diagnosticados com disgenesia tireoidiana; 4. Pesquisar o gene TSH-R numa coorte de pacientes com HC diagnosticados no programa de triagem neonatal da França. METODOLOGIA: Até o ano de 2016, 1.188 crianças foram diagnosticadas com HC e 773 estão em acompanhamento. Duzentos e dezoito crianças confirmadas com HC foram caracterizadas clinicamente através de testes de função da tireoide (TT4 e TSH), ultrassonografia e cintilografia, seguidas de dosagem de tireoglobulina. Toda a região codificantes dos genes PAX8, TSH-R, NKX2.5 e HES1 incluindo íntrons e éxons foram amplificados a partir do DNA genômico através da PCR (Reação em cadeia da Polimerase) utilizando-se técnicas padrão seguida de Sequenciamento direto. RESULTADOS: Sessenta e três pacientes foram diagnosticados com DT e 155 com glândula tópica normal. Hipoplasia representou 33,4% dos casos de DT, agenesia 19%, ectopia 27% e hemiagenesia 20,6%. Altos concentrações de TSH no teste do pezinho foram detectados no grupo das agenesias seguido das hipoplasias. Na análise genética/molecular, 31 (49,2%) dos pacientes foram identificados com o polimorfismo p.D727E em heterozigose e 4 (6,4%) em homozigose, no gene TSH-R; 4/63 pacientes tiveram o polimorfismo p.P52T em heterozigose; 14/63 apresentaram a variante polimórfica p.N181N e 2/63 apresentaram a substituição sinônima conhecida p.L645L, todos no gene TSH-R. o polimorfismo p.Glu21 foi encontrado em 54% dos pacientes e p.Gln181 encontrado em 1 paciente no gene NKX2.5. Nenhuma alteração foi encontrada no gene HES1, bem como em PAX8. CONCLUSÕES: Este é o primeiro estudo realizado na população de HC no Estado da Bahia. Análises clínicas revelaram um padrão distinto entre os subgrupos da DT quando comparados com glândula normal; 6 polimorfismos já descritos foram encontrados em dois genes candidatos. Nenhuma mutação patogênica foi encontrada. A descrição fenotípica é essencial para a correta avaliação genética e os mecanismos nela implicados, além de utilizados para predição da gravidade do HC. A identificação de novos genes ou eventos moleculares que controlam a função tireoidiana pós-natal seria de grande utilidade no esclarecimento das DT. Palavras-chave: / INTRODUCTION: Congenital hypothyroidism (CH), is the most common metabolic diseases in childhood with incidence of 1: 3000-1: 4000 newborns. A group of diseases related to alterations in the development of the thyroid, called thyroid dysgenesis (TD), is responsible for approximated 85% of all HC cases, and the majority has unknown pathogenesis. OBJECTIVES: General: clinical and genetic characterization of CH patients diagnosed with TD. Specific: 1. CH clinical characterization in individuals followed at APAE/Salvador; 2. evaluating the association between thyroid abnormalities and other abnormalities or syndromes; 3. search polymorphisms and mutations in known candidate genes for TD: PAX8, TSH-R, NKX2.5 and HES1; 4. XX METHODS: Until the year 2016, 1.188 children were diagnosed for CH and 773 were actually follow in APAE-Salvador. A continuous series of 218 children with confirmed HC were characterized clinically through thyroid function tests (TT4 and TSH), thyroid ultrasound and scintigraphy, followed by serum thyroglobulin measurement. The entire coding region of the candidate genes (PAX8, TSH-R, NKX2.5 and HES1), including exon/intron boundaries, was amplified from genomic DNA by polymerase chain reaction (PCR) using standard techniques, followed by direct sequencing. Results: Sixty-three patients were diagnosed with DT and 155 with in situ thyroid gland (ISTG). Hypoplasia represented 33,4% of all cases of DT, agenesis (19%), ectopy (27%) and hemiagenesis (20,6%). The higher screening TSH levels was in the agenetic group followed by hypoplasia. In the genetic/molecular analysis, 31 (49,2%) patients were identified with a polymorphism of TSH-R gene (p.D727E); 4/63 patients had a heterozygous p.P52T; 14/63 patients showed p.N187N polymorphic variants of the gene; and 2/63 patients presented a known p.L645L synonimous substitution. The polymorphism p.Glu21was found in 54% of patients, and p.Gln181 found in only one patient in the NKX2.5 gene. None alteration was detected in HES1 gene. CONCLUSIONS: This is the first CH population-based study in State of Bahia, Brazil. Clinical analysis revealed distinct hormonal patterns in DT subgroup when compared with ISTG, with only 6 known polymorphisms identified in few cases of TD in TSH-R, PAX8, NKX2.5 and HES1 genes. No mutation was found in a candidate genes studied. A detailed description of phenotype might be essential to target the correct genetic and mechanism implicated, and useful to predict CH severity. The identification of additional genes or molecular events controlling early postnatal thyroid function would be helpful.

INTRODUÇÃO: O hipotireoidismo congênito (HC) é uma doença, de acometimento neonatal, caracterizada por diminuição nos níveis de hormônios tireoidianos. As causas mais comuns de HC primário permanente são as alterações no desenvolvimento da glândula tireóide (disgenesia) e os defeitos de síntese dos hormônios tireóideos (disormonogênese). A determinação da etiologia do HC tem papel importante na determinação da gravidade da doença, evolução e tratamento. Essa investigação é feita através de exames como ultrassonografia e cintilografia (CINT) da tireóide. Além disso, com o conhecimento do genoma humano, diversas mutações foram descritas, sendo a investigação molecular importante para a determinação da etiologia da doença. OBJETIVOS: 1. Determinar o diagnóstico etiológico dos pacientes com HC a partir de dosagens hormonais, tireoglobulina e exames de imagem; 2. Estabelecer a importância do uso da ultrassonografia com Doppler colorido (USDC) no diagnóstico etiológico; 3. Estabelecer a importância do uso do teste do perclorato de sódio intravenoso (PSIV) no diagnóstico diferencial de HC por disormonogênese; 4. Estudar os genes PAX8 e receptor do TSH (TSHR) em pacientes com HC causado por disgenesia tireoidiana MÉTODOS: Avaliamos 40 pacientes acompanhados na APAE - São Caetano com diagnóstico de HC primário e permanente acima de 3 anos de idade. Os pacientes realizaram dosagens de T3, T4, T4 livre, TSH, tireoglobulina (TG) e anticorpo anti-TG pelo método imunofluorimétrico, além de USDC e CINT. Os pacientes com suspeita de disormonogênese foram submetidos ao teste PSIV e avaliação com otorrinolaringologista e audiometria tonal, se necessário. Os pacientes que apresentavam disgenesia tireoidiana tiveram o DNA extraído a partir de leucócitos periféricos para o estudo dos genes PAX8 e TSHR através de PCR e sequenciamento automático. RESULTADOS: Avaliamos 28 pacientes do sexo feminino e 12 do sexo masculino, após suspensão do tratamento com levotiroxina por 4 semanas. A idade média foi de 6,5 anos. O TSH médio foi 129,9 UI/mL (normal: 0,7-6,0). Os valores de T3, T4 e T4 livre variaram de 14 217 ng/dL (normal: 105-269), <1,6 15,8 g/dL (normal: 1,5-15) e < 0,3 2,7 ng/dL (normal: 0,7-1,5), respectivamente. A TG variou de <1 287 ng/dL (normal: 1,7-35). A USDC mostrou 21 pacientes com tireóide tópica (53%), 8 pacientes com tireóide ectópica (20%) e 11 pacientes com atireose (27%). Na CINT, o mapeamento identificou tireóide tópica em 20 pacientes (51%), tireóide ectópica em 13 pacientes (32%), e atireose em 7 pacientes (17%). A captação mostrou-se aumentada em 2 horas em 10 pacientes. O teste PSIV foi realizado em 9 pacientes com bócio ou glândula de tamanho normal ao USDC, cuja captação foi aumentada. Apenas um paciente apresentou vômito ao início do teste. Seis pacientes apresentaram teste positivo, considerando uma queda maior que 20%. Nenhum desses pacientes apresentava surdez neurossensorial. Encontramos discrepância entre USDC e CINT em 9 pacientes, principalmente nos casos de ectopia. A dosagem de TG auxiliou na confirmação de atireose. Os níveis mais altos de TG encontrados foram nos casos de disormonogênese causados por defeito na organificação. Assim, determinamos o diagnóstico de ectopia em 32,5% dos pacientes, hipoplasia em 20%, defeito na organificação (defeito de TPO ou THOX2) em 17,5%, atireose em 15%, defeito na TG em 7,5% e 3 casos a esclarecer (7,5%). Vinte e sete pacientes foram diagnosticados como portadores de disgenesia tireoidiana e não apresentaram mutações nos genes PAX8 e TSHR. CONCLUSÃO: Estabelecemos o diagnóstico etiológico em 37 dos 40 pacientes estudados. A USDC mostrou-se importante no diagnóstico etiológico do HC, especialmente associada à dosagem de TG. O teste PSIV mostrou-se seguro no diagnóstico diferencial do HC por disormonogênese. Não identificamos nenhuma mutação nos genes PAX8 e TSHR nos casos estudados de disgenesia / INTRODUCTION: Congenital hypothyroidism (CH) is a disease at neonatal period characterized by low thyroid hormones levels. Most common causes of primary CH are alterations at thyroid gland development (dysgenesis) and thyroid hormone synthesis defects (dyshormonogenesis). The establishment of CH etiology has important role to define the severity, evolution and treatment of the disease. This investigation is based on thyroid ultrasound and radiouptake and radionuclide imaging (RAIU). With human genome knowledge, several mutations were described, becoming molecular investigation so important to etiology definition. OBJECTIVES: 1. Establish the etiologic diagnosis of CH patients using hormonal measurements, thyroglobulin and imaging exams. 2. Establish the importance of color Doppler ultrasound (CDUS) in etiologic diagnosis. 3. Establish the importance of intravenous perchlorate sodium test in differential diagnosis of CH due to dyshormonogenesis. 4. Study PAX8 and TSH receptor (TSHR) genes in patients with CH due to thyroid dysgenesis. METHODS: We evaluated forty patients followed-up at APAE - São Caetano with primary and permanent CH diagnosis above 3 years-old. Patients performed T3, T4, free T4, TSH, thyroglobulin (TG) and anti-TG antibody using immunofluorimetric assays, besides thyroid CDUS and RAIU. Patients with thyroid dysgenesis had their DNA extracted from peripheral leukocytes to study PAX8 and TSHR genes using PCR and automatic sequencing. Patients with dyshormonogenesis suspected were submitted to intravenous perchlorate sodium test and otorhinolaryngologist and tonal audiometric evaluation, if necessary. RESULTS: We evaluated 28 female and 12 male after levothyroxine treatment off for 4 weeks. Mean age of studied patients was 6.5 years-old. Mean TSH was 129.9 UI/mL (normal: 0.7-6.0). T3, T4 and freeT4 ranged from 14 217 ng/dL (normal 105-269) , <1.6 15.8 g/dL (normal: 1.5- 15) and < 0.3 2.7 ng/dL (normal: 0.7-1.5) respectively. TG level ranged from < 1 287 ng/dL (normal 1.7-35). CDUS showed normally located thyroid in 21 patients (53%), ectopy in 8 patients (20%), and athyrosis in 11 patients (27%). At RAIU, thyroid scan identified normal located gland in 20 patients (51%), ectopy in 13 patients (32%) e athyrosis in 7 patients (17%). Two-hours uptake was elevated in ten patients. Intravenous perchlorate sodium test was performed in 9 patients with goiter or normal volume at CDUS, with normal or elevated uptake. Only one patient presented vomit. Six patients had positive test, considering more than 20% of decline. None from these patients had neurosensorial deafness. We found discrepancy between CDUS and RAIU in 9 patients, especially in ectopic cases. Thyroglobulin measurement helped to confirm athyrosis. Highest TG levels were found in dyshormonogenesis patients due to organification defects. Therefore we determined etiologic diagnosis of ectopic gland in 32,5% of patients, hypoplasia in 20%, organification defect (TPO or THOX2 defects) in 17,5%, athyrosis in 15%, thyroglobulin defect in 7,5% and three cases were undefined (7,5%). Twenty seven patients were diagnosed with thyroid dysgenesis and had no mutation in PAX8 and TSHR genes. CONCLUSION: We established the etiologic diagnosis in 37 from 40 patients here studied. CDUS was useful on etiologic diagnosis of CH, especially associated to thyroglobulin level. Intravenous perchlorate sodium test was safe and efficient in CH differential diagnosis of dyshormonogenesis. We identified no mutation in PAX8 and TSHR genes in dysgenesis cases

Orientador: Andrea Trevas Maciel-Guerra / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T22:31:37Z (GMT). No. of bitstreams: 1 Carvalho_AnneliseBarretode_M.pdf: 1757987 bytes, checksum: 457cb60680228e9be061cf287ce01cce (MD5) Previous issue date: 2009 / Resumo: A Síndrome de Turner (ST) caracteriza-se pela presença de um cromossomo X e perda parcial ou total do segundo cromossomo sexual. Suas características mais comuns são baixa estatura e disgenesia gonadal. O diagnóstico precoce é fundamental para que as pacientes possam se beneficiar de diversas condutas específicas a serem tomadas quando necessário. Objetivo: analisar uma ampla casuística de pacientes com ST e a forma do encaminhamento de pacientes com essa suspeita clínica a um serviço de referência. Sujeitos: 425 pacientes do sexo feminino encaminhadas ao Grupo Interdisciplinar de Estudos da Determinação e Diferenciação do Sexo da Faculdade de Ciências Médicas da Universidade de Campinas para investigação de ST, no período de janeiro de 1989 a outubro de 2006. Métodos: Foram analisados os seguintes dados obtidos dos prontuários clínicos: constituição cromossômica, idade, estatura (em escore z) e estadio puberal no momento do diagnóstico, origem do encaminhamento (interno ou externo ao HC) e especialidade do médico que encaminhou. Foi feita análise descritiva, comparação de pacientes com e sem ST e das pacientes com ST de acordo com a origem e a especialidade do médico, além de análise de correlação entre estatura e idade ao diagnóstico. Resultados: O diagnóstico de ST foi feito em 36,9% dos casos, com baixa freqüência de cariótipos 45,X e predominância de aberrações estruturais. A média de idade ao diagnóstico da ST foi 12,01 anos, o escore z da estatura -3,09 e havia atraso puberal em 71,4% das 63 pacientes com mais de 13 anos. Os médicos pediatras foram os que mais encaminharam pacientes com suspeita desta síndrome, e aquelas provenientes de serviços externos tiveram com maior freqüência diagnóstico confirmado de ST. Comparadas às demais, as pacientes com ST apresentavam maior déficit na estatura e maior freqüência de atraso puberal. Dentre os casos de ST, as encaminhadas do próprio HC tinham estatura mais baixa que as de origem externa, porém com idade e freqüência de atraso puberal semelhantes; as encaminhadas por pediatras eram mais jovens, porém com estatura e freqüência de atraso puberal semelhantes às daquelas encaminhadas por não pediatras. Foi encontrada correlação linear negativa significativa entre a idade ao diagnóstico e a estatura no total de pacientes com ST, porém a ausência dessa correlação nos casos encaminhados por não pediatras sugere que esse grupo de pacientes, de diagnóstico mais tardio, possa ter déficit estatural menos acentuado e atraso puberal nem sempre evidente Discussão: O presente trabalho mostrou alta freqüência de casos de ST em serviço de referência, porém com diagnóstico ainda tardio na maioria dos casos e independente de serem provenientes ou não de serviços terciários. É necessário, portanto, divulgar entre pediatras e não pediatras em todos os níveis de atenção os conhecimentos necessários ao encaminhamento e diagnóstico precoce da ST / Abstract: Turner syndrome (TS) is characterized by the presence of one X chromosome and partial or total lack of the second sex chromosome. Short stature and gonadal dysgenesis are the most frequent features. The early diagnosis is essential to allow appropriate management and institution of various therapeutic measures. Aim: To analyze a large sample of TS patients and to evaluate the referral of patients with clinical suspicion of this chromosome aberration. Subjects: 425 female patients referred to the Interdisciplinary Group of Disorders of sex development of the Faculty of medical sciences of the University of Campinas to investigate TS between January 1989 and October 2006. Methods: The following data were obtained from the medical records: chromosome constitution; age, height (zscore) and pubertal stage at diagnosis; origin of referral (from the University Hospital - UH - or outside services) and specialty of the physician who referred the patient. Descriptive analysis, followed by comparison between patients with and without TS and, among patients with TS, comparison according to origin and specialty of the physician. Correlation between age and height at diagnosis was also analyzed. Results: The diagnosis of TS was made in 36.9% of the cases, with low frequency of the 45,X karyotype and predominance of structural aberrations. Mean age of diagnosis was 12.01 years, height z score -3.09, and pubertal delay was found in 71.4% of the 63 patients aged more than 13 years. Pediatricians referred most patients, and the frequency of TS was higher among cases referred from outside services. When compared to the other patients of the sample, TS girls had shorter stature and higher frequency of pubertal delay. Among patients with TS, those referred from the UH had shorter stature but similar age and frequency of pubertal delay; those referred by pediatricians were younger, but stature and frequency of pubertal delay were similar to those referred by non-pediatricians. There was negative linear correlation between age and stature in the whole sample of girls with TS at diagnosis; however, absence of a similar correlation among girls referred by non-pediatricians suggests that in this group of patients, whose diagnosis was even more delayed, growth deficiency and puberty delay may be less evident. Discussion: The present work shows high frequency of TS in a reference service, but age at diagnosis is delayed in most cases, independent from its origin. Information about TS and the need for early diagnosis must be spread among pediatricians and non-pediatricians in all levels of health attention / Mestrado / Pediatria / Mestre em Saude da Criança e do Adolescente

Orientador: Andrea Trevas Maciel-Guerra / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T12:16:58Z (GMT). No. of bitstreams: 1 Andrade_JulianaGabrielRibeirode_M.pdf: 1561649 bytes, checksum: 09036be185a1beed5a46ce3cdf196d4e (MD5) Previous issue date: 2010 / Resumo: Introdução: A disgenesia gonadal (DG) parcial XY, caracterizada por disgenesia testicular e genitais internos e externos ambíguos em indivíduos com cariótipo 46,XY, é uma causa rara de distúrbio da diferenciação do sexo, de prognóstico ainda não completamente elucidado e etiologia ainda desconhecida. No entanto, o conhecimento a respeito de sua evolução clínica, da ocorrência de puberdade espontânea e de anomalias associadas é fundamental para definição do sexo de criação e orientação das famílias. Além disso, como genes responsáveis pela diferenciação testicular agem como promotores do crescimento, é possível haver associação dessa afecção com distúrbios do crescimento pré e pós-natal. Objetivo: Avaliar características clínicas associadas a esse distúrbio da diferenciação gonadal: antecedentes gestacionais e familiares, crescimento, puberdade espontânea e anomalias associadas. Sujeitos e Métodos: Análise retrospectiva dos dados contidos nos prontuários de 11 pacientes com DG Parcial XY criados no sexo masculino e com ao menos um dos testículos situado na bolsa escrotal. Estes pacientes haviam sido objeto de extensa avaliação clínica, histopatológica e molecular entre 1996 e 1998. Resultados: A maioria dos pacientes atingiu altura final dentro do canal de crescimento, e todos tiveram puberdade espontânea, apesar de níveis elevados de LH em alguns casos; apenas um necessitou de reposição de andrógenos para completar o desenvolvimento puberal. Havia níveis elevados de FSH e grave oligospermia; deficiência mental foi observada em três casos e hipotireoidismo em dois. Discussão: Portadores de DG parcial XY criados no sexo masculino e que tenham ao menos um testículo tópico têm bom prognóstico quanto à puberdade espontânea, embora haja possibilidade de falência secundária das células de Leydig; já o prognóstico quanto á função reprodutora é reservado. Não há indicações de que haja distúrbios do crescimento pré e pós-natal associados à disgenesia testicular. Apesar do pequeno número amostral, a maior freqüência de déficit cognitivo e hipotireoidismo observada nesta amostra indica que é necessário haver especial atenção a estas possíveis anomalias associadas / Abstract: Introduction: XY partial gonadal dysgenesis (GD), characterized by ambiguous internal and external genitália in 46,XY subjects, is a rare cause of disorder of sex development. Both prognosis and etiology are still unknown. However, knowledge regarding natural history, spontaneous puberty and associated anomalies is essential to define sex of rearing and to discuss the prognosis with the parents. In addition, as genes responsible for testicular differentiation act as growth promoters, there may be an association of this disorder with pre and postnatal growth deficiency. Aim: To evaluate the clinical features associated with this disorder of gonadal differentiation: gestational and family history, growth, spontaneous puberty and associated anomalies. Subjects and Methods: Retrospective analysis of data from the clinical files of 11 patients with XY partial GD reared as males and who had at least one testis located in the scrotum. These patients had been subject to careful clinical, histopathological and molecular evaluation between 1996 and 1998. Results: Most patients attained final height within the limits of their growth channel, and all had spontaneous pubertal development, though some had high levels of LH. Only one needed androgen replacement to complete puberty. There were high levels of FSH and severe oligospermia. Mental deficiency was observed in three cases and hypothyroidism in two. Discussion: Patients with XY partial GD with at least one topic testis have good prognosis regarding spontaneous puberty, though there is a possibility of secondary Leydig cells' failure; there is no good prognosis regarding reproductive function. There are no indications on pre or postnatal growth deficiency associated with testicular dysgenesis. Despite the small sample size, the higher frequency of cognitive deficit and hypothyroidism indicates that there must be special attention to these associated disorders / Mestrado / Saude da Criança e do Adolescente / Mestre em Saude da Criança e do Adolescente

Orientador: Marcilda Palandi de Mello / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T17:47:27Z (GMT). No. of bitstreams: 1 Fabbri_HelenaCampos_M.pdf: 5693726 bytes, checksum: f3da0b3f4f94d25ac46393e9f831a748 (MD5) Previous issue date: 2013 / Resumo: O termo Distúrbio da Diferenciação do Sexo (DDS) caracteriza-se pelo desenvolvimento genital ou gonadal incompleto ou desordenado. Os DDS com cariótipo 46,XY são caracterizados por genitália externa ambígua ou feminina, em alguns casos com gônadas disgenéticas, e presença ou ausência de derivados de Müller. Os mais frequentes são a insensibilidade androgênica, deficiência da 5-alfa-redutase tipo 2, disgenesia gonadal e DDS ovário-testicular. Vários são os genes que participam dos processos de determinação e diferenciação do sexo. Alterações no gene NR5A1, que codifica o fator de transcrição SF- 1, é responsável por diferentes fenótipos de DDS. A proteína SF-1 é expressa principalmente em tecidos esteroidogênicos (gônadas, adrenais e placenta), nas células de Sertoli, nas células de Leydig e nos ovários; é o principal regulador do metabolismo do colesterol nas células esteroidogênicas. Além disso, regula a atividade de outros genes, como os CYPs, HSD3B, StAR, SOX9, DAX1, entre outros. Na literatura são descritas alterações no gene NR5A1 associadas à DDS 46,XY, anorquia bilateral, amenorréia primária, falência ovariana precoce, hipospádia, infertilidade masculina, e alguns casos de tumores adrenais e endometrioses. Neste trabalho foi realizada a análise molecular do gene NR5A1 em 86 pacientes com DDS 46,XY, incluindo-se disgenesia gonadal completa (n = 7), disgenesia gonadal parcial (n = 18), DDS 46,XY idiopático (n = 41) e outros (n = 20). Doze alterações foram identificadas neste trabalho, sendo: sete na região codificante (p.Ser32Asn, p.Arg39Cis, p.Lis38*, p.Cis65Tir, p.L80Wfs*8, p.Cis247*, and p.Asp364Trefs*18), uma em sítio de splicing (c.1138+1G>T), duas no exon 1 nãocodificante (c.-133G>A e c.-156_-136ins18pb), três na região 5'UTR (c.-413G>A, c.- 208C>A, e c.-762C>T) e uma na região 3'UTR (c.*1286C>T). As variações aqui descritas, não foram identificadas em controles saudáveis. As análises in silico demonstraram o possível efeito deletério de cada alteração e, suas relações com o fenótipo dos indivíduos. Embora estes resultados demonstrem a importância de cada alteração para o fenótipo, haverá ainda a necessidade de se investigar os efeitos funcionais in vitro. As alterações com potencial deletério foram identificadas em maior frequência nos casos dos distúrbios da diferenciação gonadal (20%) e DDS 46,XY idiopático (22%) / Abstract: The term Disorders of Sex Differentiation (DSD) characterize incomplete or disorganized genital or gonadal development. The DSD with 46, XY karyotype may present either ambiguous or female genitalia and also dysgenetic gonads in some cases, with presence or absence of Müllerian derivatives. The most frequent are androgen insensitivity, 5-alpha-reductase type 2 deficiency, gonadal dysgenesis and ovarian-testicular DSD. There are several genes that participate in both sex determination and differentiation processes. Mutations in NR5A1 gene, which encoding SF-1, a transcription factor, are responsible for different phenotypes of DSD. The protein SF-1, which is expressed mainly in steroidogenic tissues (gonads, adrenal glands and placenta), is also express in Sertoli and Leydig cells, in the ovaries, and is the major regulator of cholesterol metabolism in steroidogenic cells. Moreover, it regulates the activity of other genes, such as CYPs, HSD3B, StAR, SOX9, DAX1, among others. The literature describes the association of changes in NR5A1 gene with 46, XY DSD, bilateral anorchia, primary amenorrhea, premature ovarian failure, hypospadias, male infertility, and some cases of adrenal tumors and endometriosis. The present work involved the molecular analysis of NR5A1 gene in 86 patients with 46, XY DSD including complete gonadal dysgenesis (n = 7), partial gonadal dysgenesis (n = 18), idiopathic 46, XY DSD (n = 41) and others (n = 20). Twelve variations had been identified: seven in the coding region (p.Ser32Asn, p.Arg39Cis, p.Lis38*, p.Cis65Tir, p.L80Wfs*8, p.Cis247*, and p.Asp364Trefs*18), one at a splice site (c.1138+1 G>T), two in the noncoding exon 1 (c.-133G>A and c.-156_-136ins18pb), three in the 5'UTR region (c.- 413G>A, c.-208C>A and c.-762C>T) and one in the 3'UTR (c.*1286C>T). The variations herein described, have not been identified in healthy controls. In silico analysis showed possible deleterious effects for each change and its correlations to individual phenotypes. Although those results demonstrate the importance of each change for the phenotype, there in vitro functional effects must be investigated. The potentially deleterious changes were identified more frequently in cases of disorders of gonadal development (20%) and idiopathic 46, XY DSD (22%) / Mestrado / Genetica Animal e Evolução / Mestra em Genética e Biologia Molecular

Orientadores: Andréa Trevas Maciel Guerra, Maricilda Palandi de Mello / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T00:38:47Z (GMT). No. of bitstreams: 1 Santos_AnaPaulados_M.pdf: 2717833 bytes, checksum: 8a3a2ff5cccd42ca60c0bc51ba3b0067 (MD5) Previous issue date: 2013 / Resumo: As disgenesias gonadais parcial (DGP) e mista (DGM) caracterizam-se por ambiguidade genital e presença de gônada disgenética associada a testículo disgenético ou dois testículos disgenéticos. Na DGP o cariótipo é 46,XY; na DGM, há mosaico 45,X/46,XY ou suas variantes (mais de duas linhagens e (ou) anomalias estruturais do cromossomo Y). Esses mosaicos podem determinar, ainda, fenótipo feminino com síndrome de Turner (ST), distúrbio da diferenciação do sexo ovotesticular (DDS OT) e esterilidade em homens com genitais normais. Independentemente do fenótipo gonadal e genital, esses indivíduos apresentam outros sinais clínicos decorrentes da linhagem 45,X, como baixa estatura, dismorfismos, anomalias cardíacas e renais e diversas afecções adquiridas. Nos últimos anos surgiram evidências de ligação entre microdeleções do Y e o mosaicismo com linhagem 45,X. Há, ainda, indicações de que a instabilidade cromossômica trazida por essas deleções possa ser mais pronunciada nas gônadas. O objetivo deste trabalho foi investigar a presença de microdeleções do Y em indivíduos com DGP e naqueles com mosaico 45,X/46,XY ou suas variantes e diferentes fenótipos. A casuística constou de 15 indivíduos com DGP e 15 com mosaicismo, dos quais a maioria apresentava DGM (11 casos). Foram analisados 38 sequence tagged sites (STS) cobrindo a região específica masculina (MSY, male specific region) em Yp, centrômero e Yq por meio da técnica de reação em cadeia da polimerase (PCR) multiplex e individual. Todos os STS investigados nos indivíduos com DGP tiveram amplificação positiva, porém havia STS de Yq ausentes em seis indivíduos com mosaicismo e DGM, dos quais dois sem alterações estruturais de Y evidentes ao cariótipo. Essas deleções se localizavam em regiões contendo genes relacionados à espermatogênese (AZFb e AZFc - azoospermia factor). A ausência de deleções nos indivíduos com DGP não confirma a hipótese de que a instabilidade desse cromossomo nas gônadas seja uma das causas dessa afecção. Por outro lado, as deleções encontradas no segundo grupo indicam, em alguns casos, associação entre alterações estruturais do Y detectáveis somente a nível molecular e o surgimento de mosaicismo. Caso sejam criados no sexo masculino e busquem procedimentos de fertilização in vitro, há risco de que esses indivíduos transmitam cromossomos Y instáveis na divisão celular / Abstract: Partial and mixed gonadal dysgenesis (PGD and MGD) are characterized by genital ambiguity and the finding of either a streak gonad and a dysgenetic testis or two dysgenetic testes. In PGD there is a 46,XY karyotype, whereas in MGD there is a 45,X/46,XY mosaic or its variants (more than two lineages and/or structural abnormalities of the Y chromosome). These mosaics are also compatible with a female phenotype and Turner syndrome, ovotesticular disorder of sex development, and infertility in men with normal external genitalia. Regardless of the gonadal and genital phenotypes, these individuals present other clinical features associated with the 45,X cell line, including short stature, dysmorphisms, cardiovascular and renal anomalies and various acquired diseases. During the last few years, evidences of a link between Y microdeletions and 45,X mosaicism have been reported. There are also indications that the instability caused by such deletions might be more significant in germ cells. The aim of this work was to investigate the presence of Y chromosome microdeletions in individuals with PGD and in those with 45,X/46,XY mosaicism or its variants and variable phenotypes. Our sample comprised 15 individuals with PGD and 15 with mosaicism, most of them with a MGD phenotype (n=11). Thirty-eight sequence tagged sites (STS) spanning the male specific region (MSY) on the Y chromosome (Yp, centromere and Yq) where analyzed by multiplex PCR and some individual reactions. All STS showed positive amplifications in the PGD group. Conversely, in the group with mosaicism, six individuals with MGD had been identified with Yq microdeletions, two of them did not have structural abnormalities of the Y chromosome recognized by routine cytogenetic analysis. The deleted STSs were located within AZFb and AZFc (Azoospermia Factor) regions, which harbor several genes responsible for spermatogenesis. Absence of deletions in individuals with PGD does not confirm the hypothesis that instability of the Y chromosome in the gonads could be one of the causes of such condition. However, deletions identified in the second group indicate that mosaicism may be associated with Y chromosome abnormalities detectable only at the molecular level. If patients with mosaicism and Y microdeletions reared as males decide to undergo in vitro fertilization, Y chromosomes which tend to be unstable during cell division may be transmitted to offspring / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

Orientador: Andréa Trevas Maciel-Guerra / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T13:17:10Z (GMT). No. of bitstreams: 1 Carvalho_AnneliseBarretode_D.pdf: 1678960 bytes, checksum: 38e5284e80abdc6135eaf891cd8d4e9a (MD5) Previous issue date: 2014 / Resumo: A síndrome de Turner (ST) tem como sinais mais frequentes baixa estatura e disgenesia gonadal; são também encontrados dismorfismos, malformações e afecções adquiridas. O fenótipo é muito variável, dificultando o estabelecimento da suspeita clínica e o diagnóstico precoce. O objetivo deste estudo foi identificar os fatores que discriminam pacientes com ST daqueles sem essa síndrome, a fim de auxiliar os médicos, particularmente pediatras, a levantar precocemente essa hipótese e solicitar o exame do cariótipo. A amostra incluiu 516 pacientes do sexo feminino com essa suspeita clínica (por baixa estatura e(ou) hipogonadismo e(ou) dismorfismos característicos) encaminhadas a serviço especializado em distúrbios da diferenciação do sexo na Universidade Estadual de Campinas, entre janeiro de 1989 e fevereiro de 2012. Foi realizado um estudo descritivo de corte transversal, com a comparação entre as pacientes com e sem ST em relação a dados de história clínica e exame físico por meio do Teste do Qui-Quadrado, Teste T de Student e análises de regressão logística univariada e múltipla. Em 186 casos (36%), a ST foi confirmada pelo cariótipo, com predominância de anomalias estruturais dos cromossomos sexuais (41,9%). Nos casos de ST, o peso ao nascimento (p= 0,024) e a estatura ao diagnóstico em escore z (p<0,001) foram menores, e o índice de massa corpórea (p<0,001), maior. Entre as pacientes de mais de 13 anos e aquelas de mais de 16 anos, foram encontrados com maior frequência atraso puberal (p<0,001) e amenorreia primária (p=0,003), respectivamente, no grupo de ST. Na análise univariada, 19 dos 26 sinais dismórficos avaliados no exame físico foram significativamente mais frequentes na ST; na multivariada, o conjunto de variáveis que permitiu discriminar os grupos com e sem ST na amostra foi, em ordem decrescente: linfedema residual de membros, pescoço alado, cúbito valgo, unhas hiperconvexas, tórax alargado, anomalias de mamilos, nevos pigmentados, hipoplasia de metacarpos, maior peso e menor estatura. A investigação de ST deve ser realizada não apenas na presença de dismorfismos típicos, mas também em fenótipos menos evidentes / Abstract: In Turner syndrome (TS) the most frequent features are short stature and gonadal dysgenesis; there may also be dysmorphic signs, congenital malformations and acquired diseases. The phenotype is highly variable, which makes it difficult to establish the clinical suspicion and to achieve early diagnosis. The aim of this study was to identify factors that discriminate patients with TS from those without this syndrome in order to help physicians, particularly pediatricians, to raise this hypothesis and request a karyotype. The sample comprised 516 female patients with this clinical suspicion (with short stature and(or) hypogonadism and(or) typical dysmorphisms) which were referred to a specialized service for disorders of sex development at State University of Campinas from January 1989 to February 2012. A descriptive transversal study was conducted, with comparison between patients with and without TS regarding clinical history and physical examination by qui-square test, t test and univariate and multiple logistic regression analyses. In 186 cases (36%) TS was confirmed by karyotyping, with predominance of structural sex chromosome abnormalities (41.9%). Patients with TS had lower birth weight (p= 0.024), lower height z-score (p<0.001) and higher body mass index (p<0.001). Among patients aged more than 13 years and those aged more than 16 years there were more frequently pubertal delay (p<0.001) and primary amenorrhea (p= 0.003), respectively, in the group of TS. In univariate analysis, 19 out of the 26 dysmorphic signs were significantly nore frequent in TS patients; in multivariate analysis, the set of variables that discriminated between patients with and without TS were, in descending order: residual lymphedema in limbs, webbed neck, cubitus valgus, hyperconvex nails, broad chest, nipple anomalies, pigmented nevi, hypoplastic metacarpals, hipoplasia de metacarpos, higher weight and lower height. Investigation of TS should be performed not only in the presence of typical dysmorphisms but also in less striking phenotypes / Doutorado / Pediatria / Doutora em Ciências

Our lab has shown that conditionally disrupting the tcfap2beta gene, responsible for the activating protein-2beta (AP-2beta) transcription factor, exclusively in the craniofacial neural crest cells, leads to anterior segment dysgenesis. Subsequent loss of the corneal endothelium results in the adherence of the iris to the corneal stroma, causing closure of the iridocorneal angle. The activating protein-2beta neural crest cell knockout (AP-2beta NCC KO) model involves a complete blockage of the both the conventional (through the trabecular meshwork) and non-conventional (uveoscleral) pathways for aqueous humor drainage, and therefore it could be used as a powerful experimental model for glaucoma. As shown by our previous work, elevated intraocular pressure (IOP) and a 35% decrease in the number of cells in the retinal ganglion cell (RGC) layer was observed in AP-2beta NCC KO mice by 2 months; 6 to 11 months sooner than other reported mouse models of glaucoma. These observations suggested that the AP-2beta NCC KO mouse could be a novel and cost-effective experimental model for glaucoma if the RGC loss occurred progressively rather than due to a congenital defect. The purpose of this research project was to investigate how the retinal ganglion cell layer and macroglial activity changes with respect to age in the AP-2beta NCC KO mutant through immunofluorescence. Specifically, it was investigated whether the loss of RGCs was progressive and due to the increased IOP caused by the blockage of the uveoscleral drainage pathway. A significant decrease in the number of RGCs was observed between P4 and P10 in the retinal periphery of both WT and AP-2beta NCC KO mice (p<0.05), which is indicative of the programmed cell death that occurs due to retinal pruning during development. No statistical difference between WT and AP-2beta NCC KO mice phenotypes was observed at postnatal day 4 (P4), suggesting that no developmental defect resulted in the significant loss of RGCs at 2 months. In all other time points investigated, while no statistical difference was found between WT and the AP-2 NCC KO mutant, a clear downward trend was present in the AP-2 NCC KO mutant retinal ganglion cell layer from P10 to P40. There was also an expression of glial fibrillary acidic protein (GFAP) by Müller cells, indicating the presence of neuroinflammation at P35 and P40. This substantiates the potential P42 starting point of neurodegeneration our lab previously observed. This was further corroborated with Müller cell-associated expression of GFAP at P35 and P40 exclusively in the AP-2beta NCC KO mouse. Overall, we have shown that the retinal damage observed in our AP-2beta NCC KO mouse is not due to a developmental defect, but rather occurs over time. Thus, this mouse model, which appears to block both the conventional and unconventional uveoscleral pathways, has a profound effect on aqueous humor drainage. As a result, the model requires relatively little time to observe an increase in IOP and subsequent RGC loss. Our findings suggest that the AP-2beta NCC KO mouse can be a novel, powerful, and extremely cost-effective experimental model for glaucoma. / Thesis / Master of Science (MSc)