Avaliação da etiologia molecular de casos de DDS 46,XX testicular/ovotesticular SRY-negativo por Sequenciamento Genômico Completo
Data
2023-12-12
Autores
Neves, Nicolle Pelicer Castro 
Orientadores
Silva, Magnus Regios Dias
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A determinação do sexo em humanos depende do equilíbrio entre as vias de sinalização celular pró-testicular e pró-ovariana, as quais atuam em paralelo e de modo antagônico. No desenvolvimento gonadal típico em indivíduos 46,XY, a expressão do gene SRY atua como gatilho para ativação do fator SOX9 e, por consequência, iniciação de uma programação molecular de determinação pró-testicular. Por outro lado, em indivíduos 46,XX, o fator SOX9 é reprimido por outros fatores, como FOXL2 (via WNT4/ß-catenina), bloqueando o desenvolvimento testicular. Mais de 40 condições distintas resultam de alterações do desenvolvimento gonadal e, atualmente, são agrupadas como Diferenças do Desenvolvimento do Sexo (DDS). Entre essas, a condição de DDS 46,XX testicular/ovotesticular (DDST/OT) compreende aqueles casos em que há desenvolvimento de testículo ou ovotestis em embriões 46,XX. Apesar da translocação do SRY ser uma explicação para parte dos casos de DDST/OT 46,XX, a maioria dos casos SRY-negativos não possuem diagnóstico molecular. Neste projeto, foi investigado a etiologia molecular de três novos casos de DDS 46,XX testicular/ovotesticular SRY-negativo, advindos de três famílias distintas, por meio de Sequenciamento Genômico Completo (WGS), buscando-se por SNPs, Indels, variações de número de cópias (CNVs), ncRNAs candidatos e da inserção de elementos transponíveis em genes candidatos com ferramentas de bioinformática e posterior confirmação por Sequenciamento de Sanger. Desse modo, o objetivo do presente projeto foi identificar novas variantes em regiões codificantes e regulatórias do genoma associadas à DDST/OT SRY-negativo, contribuindo para a elucidação do diagnóstico molecular. O WGS identificou variantes nas regiões não codificantes dos genes NR5A1, SOX9, WT1 e FOXL2NB. Essas variantes foram sequenciadas pela metodologia de Sanger, porém as variantes presentes nos genes NR5A1, WT1 e FOXL2NB não foram confirmadas por este sequenciamento, enquanto a variante no gene SOX9, também estava presente na mãe 46,XX típica, sendo este um critério de descarte. A busca por CNVs não resultou em variantes associadas com o fenótipo. Concluímos esse estudo sugerindo o uso de Sequenciamento de Nova Geração (NGS) com tecnologias mais avançadas de sequenciamento de leituras longas (long-reads).
Human sex determination depends on balancing pro-testicular and pro-ovarian cellular signaling pathways, which act in parallel and antagonistically. In typical gonadal development in individuals 46,XY, the expression of the SRY gene serves as a trigger for the activation of the SOX9 factor and, consequently, initiates molecular programming for pro-testicular determination. However, in individuals 46,XX, the SOX9 factor is repressed by other factors, such as FOXL2 (via WNT4/ß-catenin), blocking testicular development. Over 40 distinct conditions result from alterations in gonadal development and are currently grouped as Differences of Sex Development (DSD). Among these, 46,XX testicular/ovotesticular DSD (DDST/OT) comprises cases where testicular or ovotestis development occurs in 46,XX embryos. Although SRY translocation explains some 46,XX SRY-negative DDST/OT cases, most SRY-negative cases lack a molecular diagnosis. In this project investigated the molecular etiology of three new cases of SRY-negative 46,XX DDST/OT from three different families was investigated using Whole Genome Sequencing (WGS). We evaluated SNPs, Indels, copy number variations, variants in candidate ncRNAs, and transposable element insertions in candidate genes through bioinformatics methodology, with confirmation by Sanger Sequencing. This project aimed to identify new variants in the coding and regulatory regions of the genome associated with SRY-negative 46,XX testicular/ovotesticular DSD cases, contributing to the elucidation of the molecular diagnosis. WGS identified variants in the non-coding regions of the NR5A1, SOX9, WT1, and FOXL2NB genes.These variants were sequenced using the Sanger methodology; however, the variants present in the NR5A1, WT1, and FOXL2NB genes were not confirmed by this sequencing. Meanwhile, the variant in the SOX9 gene was also found in the typical 46,XX mother, an exclusion criteria. The search for copy number variations did not find variants associated with the phenotype. Therefore, we suggest that the use of Next-Generation Sequencing using long-read sequencing technologies could clarify the etiology of 46,XX DDST/OT SRY-negative .
Human sex determination depends on balancing pro-testicular and pro-ovarian cellular signaling pathways, which act in parallel and antagonistically. In typical gonadal development in individuals 46,XY, the expression of the SRY gene serves as a trigger for the activation of the SOX9 factor and, consequently, initiates molecular programming for pro-testicular determination. However, in individuals 46,XX, the SOX9 factor is repressed by other factors, such as FOXL2 (via WNT4/ß-catenin), blocking testicular development. Over 40 distinct conditions result from alterations in gonadal development and are currently grouped as Differences of Sex Development (DSD). Among these, 46,XX testicular/ovotesticular DSD (DDST/OT) comprises cases where testicular or ovotestis development occurs in 46,XX embryos. Although SRY translocation explains some 46,XX SRY-negative DDST/OT cases, most SRY-negative cases lack a molecular diagnosis. In this project investigated the molecular etiology of three new cases of SRY-negative 46,XX DDST/OT from three different families was investigated using Whole Genome Sequencing (WGS). We evaluated SNPs, Indels, copy number variations, variants in candidate ncRNAs, and transposable element insertions in candidate genes through bioinformatics methodology, with confirmation by Sanger Sequencing. This project aimed to identify new variants in the coding and regulatory regions of the genome associated with SRY-negative 46,XX testicular/ovotesticular DSD cases, contributing to the elucidation of the molecular diagnosis. WGS identified variants in the non-coding regions of the NR5A1, SOX9, WT1, and FOXL2NB genes.These variants were sequenced using the Sanger methodology; however, the variants present in the NR5A1, WT1, and FOXL2NB genes were not confirmed by this sequencing. Meanwhile, the variant in the SOX9 gene was also found in the typical 46,XX mother, an exclusion criteria. The search for copy number variations did not find variants associated with the phenotype. Therefore, we suggest that the use of Next-Generation Sequencing using long-read sequencing technologies could clarify the etiology of 46,XX DDST/OT SRY-negative .