Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2008. / Härtill 4 uppsatser.

No description available.

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

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

L’hypothyroïdie congénitale (HC), qui se traduit par une insuffisance d’hormone thyroïdienne (HT) à la naissance, est la maladie endocrinienne congénitale la plus fréquente avec une prévalence d’un cas sur 2,500 naissances vivantes. Non-traitée, cette insuffisance peut entrainer un retard de développement sévère, surtout au niveau cognitif. L’HC est le plus souvent due à un défaut lors du développement de la thyroïde (dysgénésie thyroïdienne (DT)) ou lors de la production des hormones thyroïdiennes (dyshormonogenèse (D)). La majorité des cas d’HC par dysgénésie thyroïdienne (HCDT) ont une ectopie, soit une glande mal positionnée. Contrairement aux dyshormonogenèses, qui s’expliquent fréquemment par des mutations dans les gènes responsables de la production des HT, selon un modèle autosomique récessif, les causes de l’HCDT demeurent largement inconnues. Certains arguments sont en faveur d’une prédisposition génétique (le risque relatif chez les parents de premier degré est de 40 fois supérieur à celui de la population générale) mais l’HCDT ne suit pas un modèle Mendélien: 98 % des cas sont sporadiques et 92 % des jumeaux monozygotiques sont discordants pour l’HCDT. De ce fait, nous avons suggéré une hypothèse de double-hit pour expliquer les HCDT, hypothèse combinant une prédisposition germinale (héritée ou de novo) à un évènement somatique (génétique ou épigénétique). Par le passé, nous avons étudié l’évènement somatique, mais nous n’avions pas encore étudié la prédisposition germinale. Le séquençage d’exome complet peut permettre d’identifier la cause génétique dans des formes familiales d’HC, mais aussi déterminer si les cas avec une HCDT isolée sont enrichis en variants délétères, tel qu’observé chez des patients avec une malformation cardiaque congénitale, patients qui partagent des caractéristiques similaires avec ceux atteints d’HCDT. De plus, cette technique pourrait permettre d’identifier de nouveaux gènes de prédisposition associés à l’HCDT. D’une part, nous avons séquencé l’exome d’un trio (parent-enfant) afin d’identifier la cause de l’HC dans une famille avec plusieurs enfants sévèrement atteints d’HC. D’autre part, nous avons comparé les données d’exome d’une cohorte de cas avec une HCDT isolée (HCDT non syndromique, HCDT-NS) à celles d’une cohorte contrôle, à l’aide d’une approche biaisée (gene-based burden) et non biaisée (gènes candidats). Finalement, nous avons développé le modèle de poisson-zèbre afin de pouvoir valider, in vivo, l’implication de potentiels gènes candidats, dans le développement thyroïdien. L’analyse de l’exome du trio a révélé un variant dans le gène TSHR qui co-ségrégait parfaitement avec le phénotype, et les études de minigène ont permis de montrer que ce variant intronique loin des sites d’épissage traditionnels introduisait un pseudo-exon dans la séquence du TSHR, créant ainsi un récepteur tronqué et inactif. L’analyse par comparaison de cohorte (cas-contrôle) a montré que les cas avec une HCDT-NS n’ont pas davantage de variants rares délétères comparé aux contrôles. De plus, après correction, le gene-based burden n’a pas identifié de gène candidat. Par contre, des variants rares pathogéniques ou probablement pathogéniques dans des gènes liés à l’hypothyroïdie congénitale ont été identifiés chez 42% des cas. Les études réalisées chez le poisson-zèbre sur un gène candidat, IKBKE, identifié par une analyse préliminaire de l’exome dans la cohorte de cas, confirme que les vaisseaux sanguins sont importants pour le bon positionnement de la glande thyroïde chez le poisson-zèbre, mais ne permet pas d’établir le rôle d’IKBKE dans la migration thyroïdienne. Nous avons d’abord montré que l’exome est une bonne technique pour identifier la cause de l’HC dans une famille avec plusieurs enfants atteints. Toutefois, une connaissance approfondie de la maladie et des isoformes du gène d’intérêt s’est avérée essentielle afin de bien analyser les données d’exome. Ensuite, nos résultats suggèrent que les cas avec une HCDT-NS n’ont pas davantage de variants délétères que les contrôles et que l’exome complet n’est pas suffisant pour identifier des gènes de prédisposition. Le séquençage du génome est peut-être nécessaire pour trouver une prédisposition génétique à l’HCDT-NS. Par contre, il est aussi possible que la génétique ne joue pas un rôle majeur dans les dysgénésies thyroïdiennes. Finalement, nous avons validé que le poisson-zèbre est un bon modèle pour étudier le développement de la thyroïde. / Congenital hypothyroidism (CH) is a disorder with a prevalence of one in 2,500 live births. CH can lead to severe intellectual disability if left untreated. It is most commonly caused by a defect during thyroid development (thyroid dysgenesis), which results in an ectopic gland in the majority of cases. A defect in thyroid hormone production (dyshormonogenesis) is the second most common cause of CH. In contrast to dyshormonogenesis, which generally has an identified cause and follows a Mendelian mode of inheritance, the cause of CHTD remains mostly unknown. CHTD is generally sporadic (98%) and has a high discordance rate (92%) between monozygotic twins. However, first-degree relatives are affected more often than by chance alone (40x) and there is an ethnic and female predominance. We thus hypothesized that CHTD is a disorder caused by two events, one germinal (a necessary but not sufficient predisposing factor) becoming pathogenic only if a second genetic or epigenetic event occurs at the somatic level. Whole exome sequencing (WES) can allow for identification of the genetic cause of CHTD in familial forms, but may also reveal if non-syndromic CHTD (NS-CHTD) cases are enriched in rare protein-altering variant, as seen in congenital heart malformations, a developmental defect that shares several characteristics with CHTD. Moreover, it might also identify new predisposing genes. First, we performed WES on a trio (parent-child) in a family with several siblings affected with severe CH. Second, we compared WES data of a NS-CHTD cohort with data from a control cohort, using a gene-based burden (unbiased) approach and a candidate gene (biased) approach to evaluate whether WES analysis allows to identify new predisposing genes in a well-characterized cohort. Finally, we developed the zebrafish model to test the roles of candidate genes, that will be identified by WES, in thyroid development. We first identified a variant in TSHR that segregated perfectly with the phenotype in the family with CHTD and a mini gene assay showed that this deep intronic variant induced a pseudo-exon, leading to a truncated protein missing the transmembrane domain, thus an inactive TSH receptor. Next, we found that NS-CHTD cases are not enriched in rare protein-altering variants and gene-base burden analysis did not identify novel candidate genes. However, WES data revealed pathogenic or likely pathogenic variants in CH-related genes in 42% of the NS-CHTD cases. Finally, zebrafish is a good model to study thyroid development and our results on IKBKE confirm the importance of vessels in thyroid positioning, but not its role in thyroid migration. First, we showed that WES analysis is a good tool to identify the causative variant in a family with several siblings affected by CH. However, the interpretation of the exome analysis required knowledge of the expression of the relevant isoforms and of the biology of the disease. Second, while a gene-based burden test, using WES data from a well-characterised NS-CHTD cohort, did not identified new predisposing genes, it identified pathogenic or likely pathogenic variants in 42% of the NS-CHTD cases. Whole genome sequencing might be required to identify the genetic causes in NS-CHTD. However, our result may indicate that genetics does not play a major role in thyroid dysgenesis. Finally, we have established that zebrafish is a good model to study thyroid development and may help, in the future, identify pathways implicated in this process.

L’hypothyroïdie congénitale par dysgénésie thyroïdienne (HCDT) est la condition endocrinienne néonatale la plus fréquemment rencontrée, avec une incidence d’un cas sur 4000 naissances vivantes. L’HCDT comprend toutes les anomalies du développement de la thyroïde. Parmi ces anomalies, le diagnostic le plus fréquent est l’ectopie thyroïdienne (~ 50% des cas). L’HCDT est fréquemment associée à un déficit sévère en hormones thyroïdiennes (hypothyroïdisme) pouvant conduire à un retard mental sévère si non traitée. Le programme de dépistage néonatal assure un diagnostic et un traitement précoce par hormones thyroïdiennes. Cependant, même avec un traitement précoce (en moyenne à 9 jours de vie), un retard de développement est toujours observé, surtout dans les cas les plus sévères (c.-à-d., perte de 10 points de QI). Bien que des cas familiaux soient rapportés (2% des cas), l’HCTD est essentiellement considérée comme une entité sporadique. De plus, plus de 92% des jumeaux monozygotiques sont discordants pour les dysgénésies thyroïdiennes et une prédominance féminine est rapportée (spécialement dans le cas d’ectopies thyroïdiennes), ces deux observations étant clairement incompatible avec un mode de transmission héréditaire mendélien. Il est donc cohérent de constater que des mutations germinales dans les facteurs de transcription thyroïdiens connus (NKX2.1, PAX8, FOXE1, and NKX2.5) ont été identifiées dans seulement 3% des cas sporadiques testés et furent, de plus, exclues lors d’analyse d’association dans certaines familles multiplex. Collectivement, ces données suggèrent que des mécanismes non mendéliens sont à l’origine de la majorité des cas de dysgénésie thyroïdienne. Parmi ces mécanismes, nous devons considérer des modifications épigénétiques, des mutations somatiques précoces (au stade du bourgeon thyroïdien lors des premiers stades de l’embryogenèse) ou des défauts développementaux stochastiques (c.-à-d., accumulation aléatoire de mutations germinales ou somatiques). Voilà pourquoi nous proposons un modèle «2 hits » combinant des mutations (épi)génétiques germinales et somatiques; ce modèle étant compatible avec le manque de transmission familial observé dans la majorité des cas d’HCDT. Dans cette thèse, nous avons déterminé si des variations somatiques (épi)génétiques sont associées à l’HCTD via une approche génomique et une approche gène candidat. Notre approche génomique a révélé que les thyroïdes ectopiques ont un profil d’expression différent des thyroïdes eutopiques (contrôles) et que ce profil d’expression est enrichi en gènes de la voie de signalisation Wnt. La voie des Wnt est cruciale pour la migration cellulaire et pour le développement de plusieurs organes dérivés de l’endoderme (p.ex. le pancréas). De plus, le rôle de la voie des Wnt dans la morphogénèse thyroïdienne est supporté par de récentes études sur le poisson-zèbre qui montrent des anomalies du développement thyroïdien lors de la perturbation de la voie des Wnt durant différentes étapes de l’organogénèse. Par conséquent, l’implication de la voie des Wnt dans l’étiologie de la dysgénésie thyroïdienne est biologiquement plausible. Une trouvaille inattendue de notre approche génomique fut de constater que la calcitonine était exprimée autant dans les thyroïdes ectopiques que dans les thyroïdes eutopiques (contrôles). Cette trouvaille remet en doute un dogme de l’embryologie de la thyroïde voulant que les cellules sécrétant la calcitonine (cellules C) proviennent exclusivement d’une structure extrathyroïdienne (les corps ultimobranchiaux) fusionnant seulement avec la thyroïde en fin de développement, lorsque la thyroïde a atteint son emplacement anatomique définitif. Notre approche gène candidat ne démontra aucune différence épigénétique (c.-à-d. de profil de méthylation) entre thyroïdes ectopiques et eutopiques, mais elle révéla la présence d’une région différentiellement méthylée (RDM) entre thyroïdes et leucocytes dans le promoteur de FOXE1. Le rôle crucial de FOXE1 dans la migration thyroïdienne lors du développement est connu et démontré dans le modèle murin. Nous avons démontré in vivo et in vitro que le statut de méthylation de cette RDM est corrélé avec l’expression de FOXE1 dans les tissus non tumoraux (c.-à-d., thyroïdes et leucocytes). Fort de ces résultats et sachant que les RDMs sont de potentiels points chauds de variations (épi)génétiques, nous avons lancé une étude cas-contrôles afin de déterminer si des variants génétiques rares localisés dans cette RDM sont associés à la dysgénésie thyroïdienne. Tous ces résultats générés lors de mes études doctorales ont dévoilé de nouveaux mécanismes pouvant expliquer la pathogenèse de la dysgénésie thyroïdienne, condition dont l’étiologie reste toujours une énigme. Ces résultats ouvrent aussi plusieurs champs de recherche prometteurs et vont aider à mieux comprendre tant les causes des dysgénésies thyroïdiennes que le développement embryonnaire normal de la thyroïde chez l’homme. / Congenital hypothyroidism from thyroid dysgenesis (CHTD) is the most common congenital endocrine disorder with an incidence of 1 in 4,000 live births. CHTD includes multiple abnormalities in thyroid gland development. Among them, the most common diagnostic category is thyroid ectopy (~ 50 % of cases). CHTD is frequently associated with a severe deficiency in thyroid hormones (hypothyroidism), which can lead to severe mental retardation if left untreated. The newborn biochemical screening program insures the rapid institution of thyroid hormone replacement therapy. Even with early treatment (on average at 9 d), subtle developmental delay is still be observed in severe cases (i.e., IQ loss of 10 points). Although there have been some reports of familial occurrence (in 2% of the cases), CHTD is mainly considered as a sporadic entity. Furthermore, monozygotic (MZ) twins show a high discordance rate (92%) for thyroid dysgenesis and female predominance is observed in thyroid dysgenesis (especially thyroid ectopy), these two observations being incompatible with simple Mendelian inheritance. In addition, germline mutations in the thyroid related transcription factors NKX2.1, PAX8, FOXE1, and NKX2.5 have been identified in only 3% of sporadic cases and linkage analysis has excluded these genes in some multiplex families with CHTD. Collectively, these data point to the involvement of non-Mendelian mechanisms in the etiology of the majority of cases of thyroid dysgenesis. Among the plausible mechanisms are epigenetic modifications, somatic mutations occurring in the thyroid bud early during embryogenesis, or stochastic developmental events. Hence, we proposed a two-hit model combining germline and somatic (epi)genetic variations that can explain the lack of clear familial transmission of CTHD. In this present thesis, we assessed the role of somatic (epi)genetic variations in the pathogenesis of thyroid dysgenesis via a genome-wide as well as a candidate gene approach. Our genome wide approach revealed that ectopic thyroids show a differential gene expression compared to that of normal thyroids, with enrichment for the Wnt signalling pathway. The Wnt signalling pathway is crucial for cell migration and for the development of several endoderm-derived organs (e.g., pancreas). Moreover, a role of Wnt signalling in thyroid organogenesis was further supported by recent zebrafish studies which showed thyroid abnormalities resulting from the disruption of the Wnt pathway during different steps of organogenesis. Thus, Wnt pathway involvement in the etiology of thyroid ectopy is biologically plausible. An unexpected finding of our genome-wide gene expression analysis of ectopic thyroids was that they express calcitonin similar to normally located (orthotopic) thyroids. Such a finding, although in contradiction with our current knowledge of the embryological development of the thyroid attributes C cell origins to extrathyroidal structures (ultimobrachial bodies) upon fusion with a fully-formed, normally situated gland. Using a candidate gene approach, we were unable to demonstrate any differences in the methylation profile between ectopic and eutopic thyroids, but nevertheless we documented the presence of a differentially methylated region (DMR) between thyroids and leukocytes in the promoter of FOXE1, a gene encoding the only thyroid related transcription factor known to play a crucial role in regulating the migration of the thyroid precursors during development as shown by animal studies. We demonstrated by in vivo and in vitro studies that the methylation status of this DMR is correlated with differential expression of FOXE1 in non-tumoral tissues (thyroids and leukocytes). Knowing that DMRs are hotspots for epi(genetic) variations, its screening among CTHD patients is justifiable in our search for a molecular basis of thyroid dysgenesis, currently underway in a case-control study. The results generated during my graduate studies represent unique and novel mechanisms underlying the pathogenesis of CHTD, the etiology of which is still an enigma. They also paved the way for many future studies that will aid in better understanding both the normal and pathogenic development of the thyroid gland.