Avaliação de elementos regulatórios e domínios funcionais de SRY na etiologia molecular da disgenesia gonadal 46,XY utilizando diferentes metodologias citogenômicas e moleculares
Data
2024-06-27
Autores
Ferreira, Karen Rodrigues 
Orientadores
Silva, Magnus Régios Dias da
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A determinação típica do sexo durante a gestação depende da presença ou ausência do cromossomo (chr) Y, que abriga o gene SRY (Yp11.2), o qual está localizado na fita antissenso e atua como um fator-gatilho para determinação molecular pró-testicular. Variantes patogênicas no SRY estão geralmente associadas à disgenesia gonadal (DG) 46,XY. Tem ficado evidente a importância de se investigar os elementos de regulação da expressão gênica em regiões não codificantes. Por isso, o presente trabalho rastreou variantes e rearranjos estruturais no locus gênico do SRY e regiões flanqueadoras em um caso esporádico e um caso familiar de DG 46,XY na forma completa (DGC), parcial (DGP) ou mista (DGM) acompanhados no Ambulatório de Desenvolvimento do Hospital São Paulo da Universidade Federal de São Paulo. O caso #1 é esporádico e corresponde a um indivíduo com gônada displásica e genitália atípica. O cariótipo por bandamento G no sangue periférico revelou duas linhagens celulares: 45,X,psu dic(22;Y)(p13;p?11.32)[32]/45,X[8]. A reação em cadeia da polimerase (PCR) seguida do sequenciamento do tipo Sanger demonstrou a presença do gene SRY sem variantes. O whole genome sequecing (WGS) foi utilizado para auxiliar na determinação dos pontos de quebra da translocação. Contudo, não foi possível a avaliação da região do rearranjo cromossômico. O cariótipo sugeriu que o ponto de quebra da translocação é mais telomérico em relação ao SRY. Perda de segmentos genômicos a jusante do SRY que se estendem até a banda Yp11.32 já foram associadas a casos de DGP 46,XY. Por isso, realizamos a técnica de hibridização in situ fluorescente (FISH) com sonda para o gene SHOX em sangue periférico para estimar o tamanho da perda da banda Yp11.32. No entanto, houve falha na hibridização das sondas. Já FISH em tecido testicular evidenciou a presença do SHOX, demonstrando que o ponto de quebra está a mais de 2 Mb em região mais telomérica que o SRY. A técnica de mapeamento óptico do genoma (OGM) foi também realizada para auxiliar na investigação dos segmentos genômicos envolvidos com a translocação. Embora tenha corroborado a presença de uma população de células 45,X, o OGM não identificou a translocação Y;22 observada pelo cariótipo. A partir desses dados, o caso #1 foi reclassificado como DGM 45,X/46,XY, tendo a haploinsuficiência do mosaicismo como etiologia molecular. O caso #2 é familiar, sendo que o probando #2.1 apresenta DGC 46,XY, seu meio-irmão #2.2 apresenta desenvolvimento testicular típico e seu sobrinho #2.3 (filho de #2.2) tem DGP 46,XY. Apesar da variabilidade fenotípica, dados anteriores demonstraram que os três membros estudados dessa família apresentam duplicação de 17 pb no gene SRY, a qual leva à variação no domínio C-terminal da proteína SRY (p.Tyr198CysfsX18). Investigamos se variantes em outros genes relacionados a diferenças de desenvolvimento do sexo (DDS) poderiam explicar essa variabilidade por WGS. Não foram encontradas outras variantes patogênicas que segregam com o fenótipo. Para investigar o efeito dessa variante sobre a atividade do SRY, realizamos um ensaio dual-luciferase para fatores de transcrição, porém a resposta de um dos controles do experimento não foi como esperada.
The typical determination of sex during gestation depends on the presence or absence of the Y chromosome (chr), which harbors the SRY gene (Yp11.2). This gene is located on the antisense strand and acts as a trigger factor for pro-testicular molecular determination. Pathogenic variants in SRY are generally associated with 46,XY gonadal dysgenesis (GD). The importance of investigating gene expression regulatory elements in non-coding regions has become evident. Therefore, this project screened for variants and structural rearrangements in the SRY gene locus and flanking regions in a sporadic and familial case of 46,XY GD in its complete (CGD), partial (PGD), or mixed (MGD) forms followed at the Ambulatório do Hospital São Paulo da Universidade Federal de São Paulo. Case #1 is sporadic and corresponds to an individual with dysplastic gonad and atypical genitalia. The karyotype from G banding in peripheral blood revealed two cell lines: 45,X, psu dic(22;Y)(p13;p?11.32)[32]/45,X[8]. Polymerase chain reaction (PCR) followed by Sanger sequencing demonstrated the presence of the SRY gene without variants. Whole genome sequencing (WGS) was used to determine the translocation breakpoints. However, it was not possible to evaluate the region of the chromosomal rearrangement. The karyotype suggested that the translocation breakpoint is more telomeric than the SRY. Loss of genomic segments downstream of SRY extending to the Yp11.32 band has already been associated with cases of 46,XY PGD. Therefore, we performed fluorescence in situ hybridization (FISH) with a probe for the SHOX gene in peripheral blood to estimate the size of the Yp11.32 band loss. However, probe hybridization failed. FISH was also applied to histological sections of the gonad of case #1 to investigate whether the proportion of the two cell lines corresponds to that in the peripheral blood. FISH in testicular tissue evidenced the presence of SHOX, demonstrating that the breakpoint is more than 2 Mb in a more telomeric region than the SRY. Optical genome mapping (OGM) was also performed to investigate the genomic segments involved in the translocation. Although it corroborated the presence of a population of 45,X cells, OGM did not identify the Y;22 translocation observed by karyotype. Based on these data, case #1 was reclassified as MGD 45,X/46,XY, with mosaicism haploinsufficiency as the molecular etiology. Case #2 is familial, with proband #2.1 presenting CGD 46,XY, her half-brother #2.2 showing typical testicular development, and her nephew #2.3 (son of #2.2) having 46,XY PGD. Despite phenotypic variability, previous data demonstrated that the three studied family members have a 17 bp duplication in the SRY gene, which leads to variation in the C-terminal domain of the SRY protein (p.Tyr198CysfsX18). We investigated whether variants in other genes related to differences of sex development (DSD) could explain this variability by WGS. No other pathogenic variants segregating with the phenotype were found. To investigate the effect of this variant on SRY activity, we conducted a dual-luciferase assay for transcription factors, but the response of one of the experiment's controls was not as expected.
The typical determination of sex during gestation depends on the presence or absence of the Y chromosome (chr), which harbors the SRY gene (Yp11.2). This gene is located on the antisense strand and acts as a trigger factor for pro-testicular molecular determination. Pathogenic variants in SRY are generally associated with 46,XY gonadal dysgenesis (GD). The importance of investigating gene expression regulatory elements in non-coding regions has become evident. Therefore, this project screened for variants and structural rearrangements in the SRY gene locus and flanking regions in a sporadic and familial case of 46,XY GD in its complete (CGD), partial (PGD), or mixed (MGD) forms followed at the Ambulatório do Hospital São Paulo da Universidade Federal de São Paulo. Case #1 is sporadic and corresponds to an individual with dysplastic gonad and atypical genitalia. The karyotype from G banding in peripheral blood revealed two cell lines: 45,X, psu dic(22;Y)(p13;p?11.32)[32]/45,X[8]. Polymerase chain reaction (PCR) followed by Sanger sequencing demonstrated the presence of the SRY gene without variants. Whole genome sequencing (WGS) was used to determine the translocation breakpoints. However, it was not possible to evaluate the region of the chromosomal rearrangement. The karyotype suggested that the translocation breakpoint is more telomeric than the SRY. Loss of genomic segments downstream of SRY extending to the Yp11.32 band has already been associated with cases of 46,XY PGD. Therefore, we performed fluorescence in situ hybridization (FISH) with a probe for the SHOX gene in peripheral blood to estimate the size of the Yp11.32 band loss. However, probe hybridization failed. FISH was also applied to histological sections of the gonad of case #1 to investigate whether the proportion of the two cell lines corresponds to that in the peripheral blood. FISH in testicular tissue evidenced the presence of SHOX, demonstrating that the breakpoint is more than 2 Mb in a more telomeric region than the SRY. Optical genome mapping (OGM) was also performed to investigate the genomic segments involved in the translocation. Although it corroborated the presence of a population of 45,X cells, OGM did not identify the Y;22 translocation observed by karyotype. Based on these data, case #1 was reclassified as MGD 45,X/46,XY, with mosaicism haploinsufficiency as the molecular etiology. Case #2 is familial, with proband #2.1 presenting CGD 46,XY, her half-brother #2.2 showing typical testicular development, and her nephew #2.3 (son of #2.2) having 46,XY PGD. Despite phenotypic variability, previous data demonstrated that the three studied family members have a 17 bp duplication in the SRY gene, which leads to variation in the C-terminal domain of the SRY protein (p.Tyr198CysfsX18). We investigated whether variants in other genes related to differences of sex development (DSD) could explain this variability by WGS. No other pathogenic variants segregating with the phenotype were found. To investigate the effect of this variant on SRY activity, we conducted a dual-luciferase assay for transcription factors, but the response of one of the experiment's controls was not as expected.
Descrição
Citação
FERREIRA, Karen Rodrigues. Avaliação de elementos regulatórios e domínios funcionais de SRY na etiologia molecular da disgenesia gonadal 46,XY utilizando diferentes metodologias citogenômicas e moleculares. 2024. 74f. Trabalho de Conclusão de Curso (Graduação em Biomedicina) – Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.