Identificação de genes de predisposição ao desenvolvimento de câncer de tiroide familial não medular por análise de dados de exoma
Data
2022
Autores
Nunes, Isabela Nogueira [UNIFESP]
Orientadores
Cerutti, Janete Maria [UNIFESP]
Tipo
Trabalho de conclusão de curso de graduação
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O câncer de tiroide familial pode ser classificado em medular (CMT), associado a Neoplasia Endócrina Múltipla do tipo 2 (MEN2); ou em carcinoma familial não-medular da tiroide (FNMTC).
O FNMTC constitui de 3 a 9% de todos os cânceres da tiroide e se caracteriza pela presença de pelo menos 2 ou mais parentes de primeiro grau afetados, na ausência de fatores de predisposição ambientais. Entre os casos de FNMTC, apenas 5% ocorrem sob a forma sindrômica e os genes de predisposição associados às síndromes são bem definidos. Aproximadamente 95% dos casos de FNMTC ocorrem sob a forma não-sindrômica, caracterizam-se pela presença de pelo menos 2 parentes de primeiro grau com carcinoma papilífero da tiroide (CPT), cujos genes de suscetibilidade ainda não estão bem definidos. Mais de 150 genes candidatos e alguns loci foram identificados, mas a maioria das variantes genéticas identificadas até agora conferem pequenos incrementos no risco, e explicam apenas uma pequena proporção do agrupamento familiar. Assim, devido aos genes associados e sua função, testes genéticos ainda são pouco utilizados para a forma de FNMTC não sindrômico. Além disso, a maioria dos estudos envolvem indivíduos com ancestralidade europeia e do leste asiático.
O presente projeto teve como objetivo identificar variantes genéticas de susceptibilidade a FNMTC. Foram selecionadas 7 famílias oriundas do Rio Grande do Norte, Brasil com FNMTC e pelo menos 3 parentes de primeiro grau afetados com CPT, uma vez que a probabilidade do traço familiar é de mais de 96%. A identificação dos genes candidatos foi realizada por meio do Sequenciamento completo do Exoma (WES). As bibliotecas foram preparadas utilizando o kit SureSelect Human all Exons V6 e sequenciadas utilizando a plataforma NextSeq550 high output kit (cobertura média de 300x). Os dados foram avaliados quanto a presença de variantes em 157 genes associados a FNMTC descritos na literatura. Os arquivos Variant Call Format (VCF) foram analisados por meio da ferramenta PhenoDB (phenodb.org). As variantes foram identificadas pelo ANNOVAR e filtradas de acordo com número de reads, qualidade e a frequência (<1%) no gnomAD (Genome Aggregation Database) e ABraOM (Arquivo Brasileiro Online de Mutações). A prevalência das variantes identificadas e associadas a FNMTC nas famílias analisadas foram avaliadas nos casos de cânceres esporádicos do The Cancer Genome Atlas (TCGA), no banco de dados Catalogue of Somatic Mutations in Cancer (COSMIC v92) e no Integrative OncoGenomics (intOGen). O efeito da substituição dos aminoácidos na estrutura e função das proteínas identificadas foi analisado usando plug-ins disponíveis através da interface Ensembl Variant Effect Predictor (ensembl.org/info/docs/tools/vep).
Dos 157 genes da literatura, foram encontradas alterações em POLD1, KMT2C e SOX7. Entretanto, os dados de validação por Sequenciamento de Sanger demonstraram que as variantes analisadas nos genes KMT2C e SOX7 não segregam com o fenótipo. A análise dos dados do WES revelou novos genes candidatos (MLH3, KMT2A, PRDM1, MUTYH, PTPRZ1, RYR1, PIK3CG e WNT11) que parecem segregar com o fenótipo de FNMTC nas famílias analisadas.
Familial thyroid cancer can be classified into medullary thyroid cancer (MTC), associated with Multiple Endocrine Neoplasia Type 2 (MEN2); or familial non-medullary thyroid cancer (FNMTC). FNMTC represents 3 to 9% of all thyroid cancers and is characterized by the presence of 2 or more affected first-degree relatives, in the absence of environmental predisposing factors. Among FNMTC cases, just 5% are syndromic and the predisposition genes associated to the syndromes are well defined. About 95% of FNMTC cases are non-syndromic, characterized by the presence of 2 or more first-degree relatives affected by papillary thyroid cancer (PTC), whose susceptibility genes are not well defined. More than 150 genes and some loci have been identified, but most identified variants until now confer small increments in risk and explain just a small fraction of the family cluster. So, due to the associated genes and its function, genetic tests still underused to non-syndromic FNMTC form. Furthermore, most studies involve individuals of European and East Asian ancestry. This project aimed to identify genetic variants to FNMTC susceptibility. Seven families from Rio Grande do Norte, Brazil with FNMTC and at least 3 first-degree relatives affected by PTC were select, once the probability of familial trait is more than 96%. Candidate genes were identified using Whole Exome Sequencing (WES). Libraries were prepared using the SureSelect Human all Exons V6 kit and were sequenced using the NextSeq550 high output kit platform (300x average coverage). The data was evaluated for the presence of variants in 157 genes associated with FNMTC described in the literature. The Variant Call Format (VCF) files were analyzed using the PhenoDB tool (phenodb.org). The variants were identified by ANNOVAR and filtered according to the number of reads, quality and frequency (<1%) in gnomAD (Genome Aggregation Database) e ABraOM (Online Archive of Brazilian Mutations). The prevalence of variants identified and associated with FNMTC in the analyzed families were evaluated in cases of sporadic cancers from The Cancer Genome Atlas (TCGA), in Catalogue of Somatic Mutations in Cancer database (COSMIC v92) and in Integrative OncoGenomics platform (intOGen). The effect of amino acid substitution on the proteins were analyzed using plug-ins available through the Ensembl Variant Effect Predictor interface (ensembl.org/info/docs/tools/vep). From the 157 genes identified in the literature, alterations were found in POLD1, KMT2C and SOX7. However, the validation data from Sanger Sequencing have shown that the analyzed variants in KMT2C and SOX7 did not segregate with the phenotype. The WES data analysis revealed variants in new candidate genes (MLH3, KMT2A, PRDM1, MUTYH, PTPRZ1, RYR1, PIK3CG e WNT11) that seems to segregate with the phenotype of FNMTC in the analyzed families.
Familial thyroid cancer can be classified into medullary thyroid cancer (MTC), associated with Multiple Endocrine Neoplasia Type 2 (MEN2); or familial non-medullary thyroid cancer (FNMTC). FNMTC represents 3 to 9% of all thyroid cancers and is characterized by the presence of 2 or more affected first-degree relatives, in the absence of environmental predisposing factors. Among FNMTC cases, just 5% are syndromic and the predisposition genes associated to the syndromes are well defined. About 95% of FNMTC cases are non-syndromic, characterized by the presence of 2 or more first-degree relatives affected by papillary thyroid cancer (PTC), whose susceptibility genes are not well defined. More than 150 genes and some loci have been identified, but most identified variants until now confer small increments in risk and explain just a small fraction of the family cluster. So, due to the associated genes and its function, genetic tests still underused to non-syndromic FNMTC form. Furthermore, most studies involve individuals of European and East Asian ancestry. This project aimed to identify genetic variants to FNMTC susceptibility. Seven families from Rio Grande do Norte, Brazil with FNMTC and at least 3 first-degree relatives affected by PTC were select, once the probability of familial trait is more than 96%. Candidate genes were identified using Whole Exome Sequencing (WES). Libraries were prepared using the SureSelect Human all Exons V6 kit and were sequenced using the NextSeq550 high output kit platform (300x average coverage). The data was evaluated for the presence of variants in 157 genes associated with FNMTC described in the literature. The Variant Call Format (VCF) files were analyzed using the PhenoDB tool (phenodb.org). The variants were identified by ANNOVAR and filtered according to the number of reads, quality and frequency (<1%) in gnomAD (Genome Aggregation Database) e ABraOM (Online Archive of Brazilian Mutations). The prevalence of variants identified and associated with FNMTC in the analyzed families were evaluated in cases of sporadic cancers from The Cancer Genome Atlas (TCGA), in Catalogue of Somatic Mutations in Cancer database (COSMIC v92) and in Integrative OncoGenomics platform (intOGen). The effect of amino acid substitution on the proteins were analyzed using plug-ins available through the Ensembl Variant Effect Predictor interface (ensembl.org/info/docs/tools/vep). From the 157 genes identified in the literature, alterations were found in POLD1, KMT2C and SOX7. However, the validation data from Sanger Sequencing have shown that the analyzed variants in KMT2C and SOX7 did not segregate with the phenotype. The WES data analysis revealed variants in new candidate genes (MLH3, KMT2A, PRDM1, MUTYH, PTPRZ1, RYR1, PIK3CG e WNT11) that seems to segregate with the phenotype of FNMTC in the analyzed families.