Estudo e etiologia de doenças no âmbito da citogenética humana: Abordagem convencional e MLPA
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2021-04-19
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O genoma é o que define o indivíduo, a sua espécie e as suas características individuais. Sendo uma entidade dinâmica, está em constante evolução, com o surgimento de novos elementos e o desaparecimento de outros. O DNA está compactado e distribui-se por 23 pares de cromossomas, sendo que cada cromossoma possui genes que garantem a transmissão correta da informação genética ao longo das gerações. O cariótipo é o método mais completo quanto à análise para a caracterização dos cromossomas. As anomalias em termos do número de cópias e as alterações estruturais dos cromossomas podem provocar alterações fenotípicas e mostrar-se patogénicas para os indivíduos que as possuem. A Citogenética Clássica consiste num método de análise direta dos cromossomas, através da observação direta de metafases, que visa identificá-los, classificá-los e avaliar a sua integridade e número. O cariótipo é realizado quando existe suspeita de anomalias cromossómicas e quando existem desordens do foro sexual, múltiplas anomalias congénitas e/ou défice cognitivo, dificuldades de aprendizagem, infertilidade ou abortos de repetição, no caso de se tratar de diagnóstico pós-natal, ou quando existem anomalias ecográficas ou rastreio bioquímico alterado, no caso de se tratar de diagnóstico pré-natal. Entre setembro de 2019 e março de 2020, na Unidade de Citogenética do Centro de Genética Médica Jacinto Magalhães, foram efetuadas culturas sincronizadas de linfócitos, a partir de amostras de sangue periférico, seguidas da manipulação conveniente e da técnica de bandeamento GTL, de forma a poder observar e estudar os cromossomas dos respetivos indivíduos. As células provenientes de amostras fetais foram cultivadas até que pudessem ser devidamente manipuladas, com posterior bandeamento GTL. Em ambos os casos, a análise do cariótipo foi realizada com recurso ao microscópio e ao sistema de captura e tratamento de imagens de metafases, o Citovision, de forma a avaliar o número e estrutura dos cromossomas. Das 59 amostras analisadas, identificaram-se seis anomalias. Destas, apenas duas envolviam alterações estruturais dos cromossomas. As restantes quatro consistiam em anomalias numéricas, sendo que duas envolviam os cromossomas sexuais. Foi observada a execução da técnica Fluorescent in situ hybridization, com recurso sondas específicas para a anomalia que se pretende detetar e de acordo com o protocolo estabelecido pelo fabricante. A técnica de citogenética molecular MLPA foi aplicada a amostras que requeriam tal intervenção, seguindo o protocolo definido pelo fabricante, com o objetivo de detetar alterações do número de cópias causadoras de patologia. Foram utilizados os painéis SALSA®MLPA®ProbemixP245(MRC-Holland™) e SALSA®MLPA®ProbemixP297(MRC-Holland™). Dos 50 indivíduos estudados recorrendo a esta técnica, nove apresentaram anomalia. O painel SALSA®MLPA®ProbemixP250(MRCHolland™) foi utilizado para melhor caracterizar dois dos casos e confirmar a alteração envolvida.
The human genome is what defines an individual, his species and individual characteristics. Being a dynamic entity, it is constantly evolving, with the appearance of new elements and the disappearance of others. The DNA is compacted over 23 pairs of chromosomes, each containing genes that ensure the correct transmission of genetic traits over the generations. The karyotype analysis is the most complete method regarding the characterization of the chromosomes. Abnormalities in terms of the number and structure of the chromosomes may lead to phenotypic anomalies and prove to be pathogenic for the individuals who carry them. Classical cytogenetics consists of a method of direct analysis of chromosomes, through direct observation of metaphases, which aims to identify and classify them and evaluate their integrity and number. This analysis is performed when chromosomal abnormalities are suspected and when there are sexual disorders, multiple congenital anomalies and/or cognitive impairment, learning difficulties, infertility or recurrent abortions, regarding postnatal diagnosis, or when there are echographic anomalies or altered biochemical screening, regarding prenatal diagnosis. Between September 2019 and March 2020, at the Cytogenetics Unit of Jacinto Magalhães Medical Genetics Center, synchronized lymphocyte cultures were performed from peripheral blood samples, followed by convenient manipulation and GTL banding technique, in order to observe and study the chromosomes of the respective individuals. The cells from foetal samples were cultured until they could be properly manipulated, with subsequent GTL banding. In both cases, karyotype analysis was performed using a microscope and a system for capturing and treating metaphase images named Citovision, in order to evaluate the number and structure of chromosomes. Of the 59 samples analyzed, six anomalies were identified. Only two involved structural anomalies of chromosomes. The remaining four consisted of numerical anomalies, two of which involved the sex chromosomes. It was observed the execution of the Fluorescent technique in situ hybridization, using probes specific for the anomaly that is intended to be detected and according to the protocol established by the manufacturer. The molecular cytogenetic technique MLPA was performed using samples that required such intervention, following the protocol defined by the manufacturer, with the objective of detecting chromosomal alterations causing pathology. The panels SALSA®MLPA®ProbemixP245(MRC-Holland™) and SALSA®MLPA®ProbemixP297(MRC-Holland™) were used. Of the 50 individuals studied using this technique, nine presented with anomaly. The panel SALSA®MLPA®ProbemixP250(MRC-Holland™) was used to better characterize two of the cases and confirm the alteration involved
The human genome is what defines an individual, his species and individual characteristics. Being a dynamic entity, it is constantly evolving, with the appearance of new elements and the disappearance of others. The DNA is compacted over 23 pairs of chromosomes, each containing genes that ensure the correct transmission of genetic traits over the generations. The karyotype analysis is the most complete method regarding the characterization of the chromosomes. Abnormalities in terms of the number and structure of the chromosomes may lead to phenotypic anomalies and prove to be pathogenic for the individuals who carry them. Classical cytogenetics consists of a method of direct analysis of chromosomes, through direct observation of metaphases, which aims to identify and classify them and evaluate their integrity and number. This analysis is performed when chromosomal abnormalities are suspected and when there are sexual disorders, multiple congenital anomalies and/or cognitive impairment, learning difficulties, infertility or recurrent abortions, regarding postnatal diagnosis, or when there are echographic anomalies or altered biochemical screening, regarding prenatal diagnosis. Between September 2019 and March 2020, at the Cytogenetics Unit of Jacinto Magalhães Medical Genetics Center, synchronized lymphocyte cultures were performed from peripheral blood samples, followed by convenient manipulation and GTL banding technique, in order to observe and study the chromosomes of the respective individuals. The cells from foetal samples were cultured until they could be properly manipulated, with subsequent GTL banding. In both cases, karyotype analysis was performed using a microscope and a system for capturing and treating metaphase images named Citovision, in order to evaluate the number and structure of chromosomes. Of the 59 samples analyzed, six anomalies were identified. Only two involved structural anomalies of chromosomes. The remaining four consisted of numerical anomalies, two of which involved the sex chromosomes. It was observed the execution of the Fluorescent technique in situ hybridization, using probes specific for the anomaly that is intended to be detected and according to the protocol established by the manufacturer. The molecular cytogenetic technique MLPA was performed using samples that required such intervention, following the protocol defined by the manufacturer, with the objective of detecting chromosomal alterations causing pathology. The panels SALSA®MLPA®ProbemixP245(MRC-Holland™) and SALSA®MLPA®ProbemixP297(MRC-Holland™) were used. Of the 50 individuals studied using this technique, nine presented with anomaly. The panel SALSA®MLPA®ProbemixP250(MRC-Holland™) was used to better characterize two of the cases and confirm the alteration involved
Descrição
Este trabalho foi expressamente elaborado com vista à obtenção do grau de Mestre em Biotecnologia para as Ciências da Saúde.
Palavras-chave
Cromossomas , Citogenética