Identificação de alterações genéticas no exoma completo de pacientes com hipotireoidismo congênito por disgenesia tireoidiana
Date
2022-02-25
Authors
Brust, Ester Saraiva [UNIFESP]
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Rubió, Ileana Gabriela Sanchéz de [UNIFESP]
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Tese de doutorado
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Abstract
O hipotireoidismo congênito (HC) é um distúrbio metabólico sistêmico, onde a produção de T3 e T4 no período neonatal é insuficiente. Os pacientes com HC apresentam disormonogênese (defeitos de síntese ou secreção dos hormônios tireoidianos, afetando 10-15% dos casos) ou disgenesia tireoidiana (DT) (defeitos no desenvolvimento da tireoide, afetando 80-85% dos casos). A DT já foi relacionada a mutações nos genes HOXA3, NKX2-1, PAX8, FOXE1, HHEX, HES1, EYA1, TSHR, TUBB1, GLIS3, JAG1, NKX2-5, NTN1 e CDCA8, embora tenham sido identificadas em apenas 5% dos pacientes. Por isso, é necessário continuar investigando as bases moleculares da embriogênese tireoidiana.
Objetivo: Ampliar o estudo das bases moleculares da DT através da pesquisa de alterações gênicas com relevância clínica no exoma completo e em sequências regulatórias, utilizando sequenciamento de nova geração e análises in silico.
Métodos: A casuística deste estudo foi composta por 71 pacientes com DT, 35 destes foram submetidos ao sequenciamento do exoma. As análises foram realizadas considerando genes alterados e variantes em comum nas DTs, variantes em genes candidatos (associados ao desenvolvimento e função tireoidiana) e regulados pelos fatores de transcrição FOXE1 e PAX8, e alterações cromossômicas. Informações das variantes sobre frequências populacionais e associação com doenças foram obtidas dos bancos de dados: 1000G, ABraOM, dbSNP, ExAc, ClinVar e DGV. Para a predição do efeito das variantes foram utilizados os programas: Enrichr, FannsDB-Condel, FATHMM-MKL, HOPE, MutationAssessor, MutationTaster, PolyPhen2, String e TFBind. A confirmação de variantes de interesse e análises de genótipo em pacientes e indivíduos controles normais brasileiros foram realizados por sequenciamento Sanger.
Resultados: Observamos em todos os pacientes genes alterados associados ao desenvolvimento, morfogênese, migração e adesão celular, assim como variantes raras (MAF<1%) e deletérias compartilhadas entre as DTs. Identificamos variantes raras e deletérias em genes candidatos, das quais se destacaram TSHR_p.T574S, FGF3_p.K101N e SFRP2_p.S235X. Também identificamos 5 variantes no gene DUOX2 potencialmente prejudiciais e variantes em genes regulados por FOXE1 e PAX8, contudo, sem comprometer os motifs de ligação dos fatores de transcrição. Nas análises de genotipagem verificamos que as variantes rs1867277 e rs71369530 no gene FOXE1 foram fatores de risco para a DT (OR=2,45 (95%CI=1,69-3,55) e OR=1,78 (95%CI=1,10-2,88), respectivamente), com uma associação mais forte na presença conjunta das duas variantes (OR=2,90; 95%CI=1,24-6,72). Não identificamos CNVs ou regiões em homozigose prejudiciais. As análises de interação dos genes alterados sugerem uma possível causa poligênica da DT em nossos casos.
Conclusões: Este estudo permitiu amplamente rastrear variantes em casos de DT e identificar alterações com potencial relevância clínica. Ainda, polimorfismos no gene FOXE1 foram fatores de risco para o desenvolvimento de malformações da tireoide. Nossos resultados sugerem que genes até então não associados às DTs podem estar envolvidos na doença e, ainda, reforçam a inexistência de um padrão genético único para as causas da DT. Os modelos poligênico ou de múltiplos fatores também podem estar relacionados com as falhas de desenvolvimento da tireoide, os quais devem ser mais explorados. Não descartamos a necessidade de ensaios funcionais e até mesmo o uso de modelos animais que podem fornecer dados mais conclusivos a associação destas variantes com a DT.
Congenital hypothyroidism (CH) is a systemic metabolic disorder where the production of T3 and T4 in the neonatal period is insufficient. Patients with CH have dyshormonogenesis (defects in the synthesis or secretion of thyroid hormones, affecting 10-15% of cases) or thyroid dysgenesis (TD) (defects in thyroid development, affecting 80-85% of cases). DT has been linked to mutations in HOXA3, NKX2-1, PAX8, FOXE1, HHEX, HES1, EYA1, TSHR, TUBB1, GLIS3, JAG1, NKX2-5, NTN1 and CDCA8 genes, although they have been identified in only 5% of patients. Therefore, it is necessary to continue investigating the molecular basis of thyroid embryogenesis. Objective: To expand the molecular basis study of TD through the investigation of genetic alterations with clinical relevance in the whole exome and in regulatory sequences, using next-generation sequencing and in silico analysis. Methods: The sample of this study consisted of 71 patients with TD, 35 of whom underwent exome sequencing. The analyzes were performed considering altered genes and variants in common in TDs, variants in candidate genes (associated with thyroid development and function) and regulated by FOXE1 and PAX8 transcription factors, and chromosomal alterations. Variant information on population frequencies and disease association was obtained from the following databases: 1000G, ABraOM, dbSNP, ExAc, ClinVar and DGV. To predict the variants effect, the following programs were used: Enrichr, FannsDB-Condel, FATHMM-MKL, HOPE, MutationAssessor, MutationTaster, PolyPhen2, String and TFBind. Confirmation of variants of interest and genotype analyzes in patients and normal Brazilian controls subjects were performed by Sanger sequencing. Results: We observed in all patients altered genes associated with development, morphogenesis, migration and cell adhesion, as well as rare (MAF<1%) and deleterious variants shared between DTs. We identified rare and deleterious variants in candidate genes, of which TSHR_p.T574S, FGF3_p.K101N and SFRP2_p.S235X stood out. We also identified 5 potentially harmful variants in the DUOX2 gene and variants in genes regulated by FOXE1 and PAX8, however, without compromising the transcription factor binding motifs. In the genotyping analysis, we found that the rs1867277 and rs71369530 variants in the FOXE1 gene were risk factors for TD (OR=2.45 (95%CI=1.69-3.55) and OR=1.78 (95%CI =1.10-2.88), respectively), with a stronger association in the joint presence of the two variants (OR=2.90; 95%CI=1.24-6.72). We did not identify harmful CNVs or homozygous regions. The interaction analyzes of the altered genes suggest a possible polygenic cause of TD in our cases. Conclusions: This study made it possible to broadly screen variants in TD cases and identify changes with potential clinical relevance. Furthermore, polymorphisms in the FOXE1 gene were risk factors for the development of thyroid malformations. Our results suggest that genes not previously associated with TDs may be involved in the disease and, furthermore, reinforce the inexistence of a single genetic pattern for the causes of TD. Polygenic or multiple factor models may also be related to thyroid development failures, which should be further explored. We do not rule out the need for functional assays and even the use of animal models that can provide more conclusive data on the association of these variants with TD.
Congenital hypothyroidism (CH) is a systemic metabolic disorder where the production of T3 and T4 in the neonatal period is insufficient. Patients with CH have dyshormonogenesis (defects in the synthesis or secretion of thyroid hormones, affecting 10-15% of cases) or thyroid dysgenesis (TD) (defects in thyroid development, affecting 80-85% of cases). DT has been linked to mutations in HOXA3, NKX2-1, PAX8, FOXE1, HHEX, HES1, EYA1, TSHR, TUBB1, GLIS3, JAG1, NKX2-5, NTN1 and CDCA8 genes, although they have been identified in only 5% of patients. Therefore, it is necessary to continue investigating the molecular basis of thyroid embryogenesis. Objective: To expand the molecular basis study of TD through the investigation of genetic alterations with clinical relevance in the whole exome and in regulatory sequences, using next-generation sequencing and in silico analysis. Methods: The sample of this study consisted of 71 patients with TD, 35 of whom underwent exome sequencing. The analyzes were performed considering altered genes and variants in common in TDs, variants in candidate genes (associated with thyroid development and function) and regulated by FOXE1 and PAX8 transcription factors, and chromosomal alterations. Variant information on population frequencies and disease association was obtained from the following databases: 1000G, ABraOM, dbSNP, ExAc, ClinVar and DGV. To predict the variants effect, the following programs were used: Enrichr, FannsDB-Condel, FATHMM-MKL, HOPE, MutationAssessor, MutationTaster, PolyPhen2, String and TFBind. Confirmation of variants of interest and genotype analyzes in patients and normal Brazilian controls subjects were performed by Sanger sequencing. Results: We observed in all patients altered genes associated with development, morphogenesis, migration and cell adhesion, as well as rare (MAF<1%) and deleterious variants shared between DTs. We identified rare and deleterious variants in candidate genes, of which TSHR_p.T574S, FGF3_p.K101N and SFRP2_p.S235X stood out. We also identified 5 potentially harmful variants in the DUOX2 gene and variants in genes regulated by FOXE1 and PAX8, however, without compromising the transcription factor binding motifs. In the genotyping analysis, we found that the rs1867277 and rs71369530 variants in the FOXE1 gene were risk factors for TD (OR=2.45 (95%CI=1.69-3.55) and OR=1.78 (95%CI =1.10-2.88), respectively), with a stronger association in the joint presence of the two variants (OR=2.90; 95%CI=1.24-6.72). We did not identify harmful CNVs or homozygous regions. The interaction analyzes of the altered genes suggest a possible polygenic cause of TD in our cases. Conclusions: This study made it possible to broadly screen variants in TD cases and identify changes with potential clinical relevance. Furthermore, polymorphisms in the FOXE1 gene were risk factors for the development of thyroid malformations. Our results suggest that genes not previously associated with TDs may be involved in the disease and, furthermore, reinforce the inexistence of a single genetic pattern for the causes of TD. Polygenic or multiple factor models may also be related to thyroid development failures, which should be further explored. We do not rule out the need for functional assays and even the use of animal models that can provide more conclusive data on the association of these variants with TD.