Investigação dos mecanismos envolvidos na fromação e estabilização de cromossomos em anel, marcadores supranumerários e deleções terminais
Data
2015-04-29
Autores
Guilherme, Roberta dos Santos 
Orientadores
Melaragno, Maria Isabel de Souza Aranha
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Devido ao desenvolvimento das técnicas de citogenética molecular, entre elas as de FISH (do ingles, Fluorescent In Situ Hybridization) e array, atualmente é possível identificar e caracterizar diferentes tipos de anormalidades cromossômicas com maior resolução. As técnicas de PCR (do ingles, Polymerase Chain Reaction) e sequenciamento do DNA permitem analisar as regiões do genoma envolvidas nos rearranjos cromossômicos, nos fornecendo uma melhor compreensão da arquitetura do genoma relacionada à sua origem. Entre os rearranjos cromossômicos, as deleções terminais, os cromossomos em anel não supranumerários e os pequenos cromossomos marcadores supranumerários (sSMC) constituem tipos peculiares de cromossomos anormais, especialmente em termos de mecanismos de formação e estabilização. Neste estudo nós analisamos um total de 53 pacientes: 14 com cromossomos em anel não supranumerários, 29 com sSMC e 10 com deleção terminal 18q, com o intuito de investigar suas regiões teloméricas e seu papel na estabilização destas alterações cromossômicas. Foram realizadas as técnicas de citogenética clássica e molecular, incluindo array de alta resolução, PCR e sequenciamento para determinar a extensão dos desequilíbrios genômicos e seus pontos de quebra. Foi utilizada sonda PNA telomere para a técnica de FISH que mostrou que tanto cromossomos em anel não supranumerários quanto sSMCs podem ou não apresentar telômeros. Todos os pacientes com deleção terminal 18q analisados apresentaram sequências teloméricas repetidas adicionadas às suas extremidades terminais deletadas. O sequenciamento dos pontos de junção de seis pacientes com deleção terminal 18q mostrou a presença de sequências teloméricas (TTAGGG)n diretamente ligadas aos pontos de quebra, sugerindo que a estabilização nestes casos foi pelo mecanismo de cicatrização telomérica. Uma paciente com deleção terminal 18q apresentou um rearranjo complexo com duas regiões deletadas, uma intersticial e outra terminal, além da adição de 17 nucleotídeos, mostrando que algumas deleções terminais, quando estudadas no nível molecular, devem ser muito mais complexas do que inicialmente suposto quando estudadas por meio das técnicas de citogenética.
Due to the development of molecular cytogenetic approaches, among them FISH (Fluorescent In Situ Hybridization) and array, it is possible nowadays to identify and characterize different types of chromosome abnormalities with a higher resolution. PCR (Polymerase Chain Reaction) technique and sequencing DNA permit the analysis of the regions involved in the rearrangements, giving us a better insight into the genomic architecture related to their origin. Among the chromosome rearrangements, terminal deletions, non-supernumerary ring chromosomes and small supernumeray marker chromosomes (sSMC) constitute peculiar types of abnormal chromosomes, especially in terms of formation and stabilization mechanisms. In this study we analyzed a total of 53 patients: 14 with non-supernumerary ring chromosomes, 29 with sSMC and 10 with terminal 18q deletion, in order to investigate their telomeric regions and their role in the stabilization of these chromosome abnormalities. We performed classic and molecular cytogenetic techniques, including high resolution array, PCR, and sequencing, to determine the extent of the genomic imbalances and their breakpoints. FISH using PNA telomere probe showed that non-supernumerary ring chromosomes and sSMCs can present or not, telomeres. In all patients with terminal 18q deletion analyzed, telomeric repeated sequences were present. Breakpoint sequencing of six patients with terminal 18q deletion showed the presence of telomeric sequences (TTAGGG)n directly attached to the breakpoints, suggesting that the stabilization was achieved by the telomere healing mechanism. One patient with 18q deletion presented a complex rearrangement with two deleted regions, one interstitial and the another terminal, with additional 17 nucleotides, showing that some terminal deletions, when studied at molecular level, may be much more complex than first thought when studied by cytogenetic approaches.
Due to the development of molecular cytogenetic approaches, among them FISH (Fluorescent In Situ Hybridization) and array, it is possible nowadays to identify and characterize different types of chromosome abnormalities with a higher resolution. PCR (Polymerase Chain Reaction) technique and sequencing DNA permit the analysis of the regions involved in the rearrangements, giving us a better insight into the genomic architecture related to their origin. Among the chromosome rearrangements, terminal deletions, non-supernumerary ring chromosomes and small supernumeray marker chromosomes (sSMC) constitute peculiar types of abnormal chromosomes, especially in terms of formation and stabilization mechanisms. In this study we analyzed a total of 53 patients: 14 with non-supernumerary ring chromosomes, 29 with sSMC and 10 with terminal 18q deletion, in order to investigate their telomeric regions and their role in the stabilization of these chromosome abnormalities. We performed classic and molecular cytogenetic techniques, including high resolution array, PCR, and sequencing, to determine the extent of the genomic imbalances and their breakpoints. FISH using PNA telomere probe showed that non-supernumerary ring chromosomes and sSMCs can present or not, telomeres. In all patients with terminal 18q deletion analyzed, telomeric repeated sequences were present. Breakpoint sequencing of six patients with terminal 18q deletion showed the presence of telomeric sequences (TTAGGG)n directly attached to the breakpoints, suggesting that the stabilization was achieved by the telomere healing mechanism. One patient with 18q deletion presented a complex rearrangement with two deleted regions, one interstitial and the another terminal, with additional 17 nucleotides, showing that some terminal deletions, when studied at molecular level, may be much more complex than first thought when studied by cytogenetic approaches.
Descrição
Citação
GUILHERME, Roberta dos Santos. Investigação dos mecanismos envolvidos na fromação e estabilização de cromossomos em anel, marcadores supranumerários e deleções terminais. 2015. 167 f. Tese (Doutorado em Biologia Estrutural e Funcional) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.