Estudo molecular e funcional de genes identificados por sequenciamento paralelo em larga escala numa coorte de pacientes com hipotiroidismo congênito por disgenesias tiroidianas

• Ano de defesa: 2016
• Autor(a) principal: Kizys, Marina Malta Letro [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Disgenesia of thyroid; Congenital hypothyroidism; Exoma; Sequencing of nucleotides in large scale; Disgenesia da tireoide; Hipotireoidismo congênito; Sequenciamento de nucleotídeos em larga escala
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=3855116; https://repositorio.unifesp.br/handle/11600/46655

Congenital hypothyroidism (CH) is one of the most common endocrine disorder among newborns, with an incidence of CH is 1/1500-4000. The main cause is developmental defects on thyroid, collectively known as thyroid dysgenesis (TD). It is believed that TD results from the impaired specification, proliferation, or survival of thyroid precursor cells. Thyroid development is under control of a combined expression of a set of transcription factors such as NKX2-1, FOXE1, PAX8, and HHEX. However, just the minority of CH patients have mutations in these transcription factors. New genes derived from animal models related to the thyroid gland development have been described. So, we expanded the study of these genes in patients diagnosed with TD. In this first step, we performed the molecular screening by Sanger sequencing. Secondly, we proceeded with the molecular study through whole exome sequencing (WES) searching for new causative CH genes. In this way, we developed a pipeline of bioinformatics to both particularized and global gene analysis. Applying this pipeline, we identified some genes possibly associated with the phenotypes of the different forms of TD. We initially screened two candidate genes, dual oxidase 2 (DUOX2) and Coiled-coil domain-containing protein 114 (CCDC114) in additional patients. Besides that, their respective mutants were functionally studied in vitro and in vivo (zebrafish). Using WES and Sanger, we identified variations in CCDC114 gene in father and his son, and also in 5 of 6 sporadic patients with hemiagenesis. Functional in silico analysis suggested that 3 of 6 variations were deleterious by at least two different prediction bioinformatics program. Through in vivo functional analysis, we conducted experiments with increasing dose of morpholino experiments in zebrafish embryos that showed a phenotype of thyroid and cardiac positioning randomly altered, not being possible to correlate the deleterious variants in CCDC114 with loss-of-function of its ortholog in zebrafish. So, despite the absence of a particular thyroid phenotype in zebrafish, we observed an association of CCDC114 mutations with hemiagenesis in humans. Using the same approach, we identified 15 variations in the DUOX2 gene in patients with ectopia, being seven new ones: G201E, L264CfsX57, P609S, M650T, E810X, M822V, and E1017G. All children carrying DUOX2 mutations segregated with CH, which was confirmed by high thyroid-stimulating hormone (TSH) levels at neonatal screening, or at postnatal confirmatory diagnosis. The thyroid scintigraphy confirmed ectopia in all affected. These mutations are heterozygous compounds with functional polymorphic variants previously related to transient or permanent hypothyroidism. We verified, in vitro, that 3 of 7 abolished the translocation of the DUOX2/DUOXA2 complex to the cell membrane and impaired the DUOX2 and thyroperoxidase necessary for the thyroid hormone synthesis. Our data reinforce the utility of the WES approach to cases with complex etiopathogenesis. Part of genes appointed by WES were unexpected findings, based on their primary function of these genes already reported in the literature, as DUOX2, in which, besides its established role in hormonogenesis, it seems to be involved in thyroid ontogenesis.