Estudos Citogenéticos em indivíduos em idade pediátrica com suspeita clínica de Síndrome de Williams: abordagem por MLPA
Data
2020-02-03
Autores
Título da revista
ISSN da revista
Título do Volume
Editora
Resumo
A obtenção de melhores preparações cromossómicas e a descoberta do número de cromossomas da espécie humana permitiu o desenvolvimento da citogenética clínica, especialmente após a descoberta de que certas doenças resultam de alterações na estrutura e/ou número dos cromossomas. O desenvolvimento das técnicas de bandeamento permitiu a identificação dos cromossomas assim como a sua análise. Apesar destes desenvolvimentos, ainda existiam limitações e foram criadas técnicas com resoluções superiores para as colmatar. Para tal, foram desenvolvidas técnicas citogenéticas moleculares que oferecem uma melhor resolução e a possibilidade de deteção de anomalias que antes não eram possíveis de detetar. Técnicas como a amplificação da sonda dependente da ligação multiplex (MLPA) que permite analisar até 50 sequências de DNA numa única reação e detetar variações no número de cópias (CNVs) de genes específicos, sendo por isso de interesse para detetar síndromes causadas por CNVs. A síndrome de Williams-Beuren (WBS) é uma dessas síndromes. É causada por uma microdeleção no braço longo do cromossoma 7 em 7q11.23. Os pacientes são afetados por um fenótipo muito característico que envolve dismorfia facial, atraso mental, malformação cardíaca congénita e um perfil psicológico específico. Ao longo deste estágio foi possível adquirir competências experimentais em técnicas de citogenética convencional e molecular através da observação e prática das mesmas. Foram tratadas mais de 400 amostras de sangue periférico e analisadas 25 amostras de sangue e duas de líquido amniótico com vista a obtenção do cariótipo. Alguns casos de interesse incluem, uma síndrome de Edwards, uma síndrome de Klinefelter, uma translocação Robertsoniana, uma inversão pericêntrica do cromossoma 1 e uma trissomia do cromossoma 16 em mosaico de baixa expressão que tinha sido detetada por aCGH. Foi implementada uma nova análise com a técnica de MLPA e o painel P029 B1-0315, assim como um novo software de análise (Coffalyser.Net) que mostrou excelentes resultados. Com esta análise foi possível caracterizar pacientes com WBS, oferecendo um teste genético rápido para confirmação da suspeita clínica assim como uma técnica mais simples para detetar a extensão de duplicações da região 7q11.23.
Due to the discovery of the number of human chromosomes as well as obtaining better chromosome preparations, it became possible for clinical cytogenetics to evolve, especially when it was found that changes in structure and/or number of chromosomes could cause diseases. The banding techniques allowed the identification and analyses of chromosomes. Despite these advancements there were limitations to the older techniques and new techniques were created with increased resolution to overtake those limitations. Molecular cytogenetic techniques were developed, allowing superior resolutions and the ability to detect anomalies that were previously undetectable. One of these techniques is called multiplex ligationdependent probe amplification (MLPA). This technique allows the analysis of up to 50 DNA sequences in a single reaction and can detect copy number variations (CNVs) of specific genes. This technique is especially useful for detection of syndromes caused by CNVs. Williams-Beuren syndrome (WBS) is one of those syndromes. It is caused by a microdeletion in the long arm of the chromosome 7, in 7q11.23. WBS patients have a very characteristic phenotype which includes facial dysmorphism, mental retardation, congenital heart defects and a specific psychological profile. During this MSc internship, experience in conventional and molecular cytogenetic techniques was acquired through observation and practice. There were more than 400 samples of blood treated and there were 27 samples of blood and amniotic fluid analyzed, in order to obtain their karyotype. The most interesting cases studied include, Edwards syndrome, Klinefelter syndrome, a Robertsonian translocation, a pericentric inversion in chromosome 1 and a low expression mosaic chromosome 16 trisomy that was detected by aCGH. A new analysis with the MLPA technique was implemented, panel P029 B1-0315, as well as a new analysis software (Coffalyser.Net), which showed excellent results. This analysis allowed the distinction of WBS patients from normal individuals, offering a quick genetic test to confirm the clinical suspicion of WBS and is a simpler technique to determine the extension of duplications in the 7q11.23 region.
Due to the discovery of the number of human chromosomes as well as obtaining better chromosome preparations, it became possible for clinical cytogenetics to evolve, especially when it was found that changes in structure and/or number of chromosomes could cause diseases. The banding techniques allowed the identification and analyses of chromosomes. Despite these advancements there were limitations to the older techniques and new techniques were created with increased resolution to overtake those limitations. Molecular cytogenetic techniques were developed, allowing superior resolutions and the ability to detect anomalies that were previously undetectable. One of these techniques is called multiplex ligationdependent probe amplification (MLPA). This technique allows the analysis of up to 50 DNA sequences in a single reaction and can detect copy number variations (CNVs) of specific genes. This technique is especially useful for detection of syndromes caused by CNVs. Williams-Beuren syndrome (WBS) is one of those syndromes. It is caused by a microdeletion in the long arm of the chromosome 7, in 7q11.23. WBS patients have a very characteristic phenotype which includes facial dysmorphism, mental retardation, congenital heart defects and a specific psychological profile. During this MSc internship, experience in conventional and molecular cytogenetic techniques was acquired through observation and practice. There were more than 400 samples of blood treated and there were 27 samples of blood and amniotic fluid analyzed, in order to obtain their karyotype. The most interesting cases studied include, Edwards syndrome, Klinefelter syndrome, a Robertsonian translocation, a pericentric inversion in chromosome 1 and a low expression mosaic chromosome 16 trisomy that was detected by aCGH. A new analysis with the MLPA technique was implemented, panel P029 B1-0315, as well as a new analysis software (Coffalyser.Net), which showed excellent results. This analysis allowed the distinction of WBS patients from normal individuals, offering a quick genetic test to confirm the clinical suspicion of WBS and is a simpler technique to determine the extension of duplications in the 7q11.23 region.
Descrição
Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Citogenética clínica , cariótipo