Investigação de mecanismos de formação de rearranjos intracromossômicos e sua relação com o fenótipo
Data
2022
Autores
Santos, Bruna Ferreira Burssed dos 
Orientadores
Melaragno, Maria Isabel de Souza Aranha
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: As alterações cromossômicas estruturais decorrem de quebras e rearranjos nos cromossomos, podendo alterar a quantidade de material genético presente na célula. Os rearranjos intracromossômicos envolvem apenas um cromossomo, podendo haver inversões, cromossomos em anel e rearranjos intrabraço, como inserções intrabraço e rearranjos do tipo inv dup del, esses caracterizados por uma duplicação invertida associada com uma deleção terminal. Diversos mecanismos têm sido descritos para explicar a formação de rearranjos cromossômicos, dentre eles a Recombinação Homóloga Não-Alélica (NAHR) e a União de Extremidade Não-Homóloga (NHEJ). A análise da sequência do DNA nos pontos de quebra das alterações cromossômicas pode revelar seus mecanismos de formação bem como apontar fatores de risco pois pode expor características relacionadas a mecanismos de reparo do DNA que moldam os rearranjos cromossômicos. Objetivo: Estudar rearranjos cromossômicos raros envolvendo simultaneamente deleções e duplicações intracromossômicas. Métodos: Seis pacientes com duplicações e deleções intracromossômicas foram investigados por meio de cariotipagem, array genômico de catálogo e customizado, hibridação in situ fluorescente (FISH), sequenciamento do genoma completo e sequenciamento de Sanger. Resultados: Três pacientes tiveram seus rearranjos completamente caracterizados e seus pontos de quebra sequenciados. Um deles apresentou um rearranjo mais complexo do que o esperado formado por mecanismo de replicação que resultou na inserção de um fragmento mais distal a um dos pontos de quebra e que deveria estar deletado mas que se encontrava no ponto de junção. Dois deles apresentaram rearranjos do tipo inv dup del com um espaçador entre as duplicações e presença de micro-homologia, sendo proposto o mecanismo Fold-back para suas formações. Outro paciente teve parte de seu rearranjo complexo caracterizado apesar da discordância entre arrays devido à presença de Low Copy Repeats (LCRs). Nos dois pacientes com cromossomo em anel foi revelada a presença de uma duplicação seguida por uma deleção terminal. Conclusões: A realização de diversas técnicas permitiu uma análise detalhada dos diferentes rearranjos dos pacientes, o que possibilitou a inferência de seus possíveis mecanismos de formação e a realização da correlação dos desequilíbrios cromossômicos com os fenótipos dos pacientes.
Background: Structural chromosomal alterations derive from breaks and rearrangements in the chromosomes and can alter the amount of genetic material present in the cell. Intrachromosomal rearrangements involve only one chromosome and include inversions, ring chromosomes and intra-arm rearrangements, such as intra-arm insertions and inv dup del rearrangements, which are characterized by an inverted duplication associated with a terminal deletion. Various mechanisms have been described to explain their formation, such as Non-Allelic Homologous Recombination (NAHR) and Non-Homologous End-Joining (NHEJ). The analysis of the DNA sequence in the breakpoints of the chromosomal alterations can reveal their mechanisms of formation as well as point out risk factors as it can expose characteristics related to DNA repair mechanisms that shape the chromosomal. rearrangements. Objective: To study rare chromosomal rearrangements involving intrachromosomal duplications and deletions. Methods: Six patients with intrachromosomal duplications and deletions were investigated by karyotyping, catalog and custom genomic array, fluorescent in situ hybridization (FISH), whole genome sequencing, and Sanger sequencing. Results: Three patients had their rearrangements completely characterized and their breakpoints sequenced. One of them presented a more complex rearrangement than expected formed by a replication mechanism which leads to the insertion of a fragment more distal to one of the breakpoints, which was supposed to be deleted but was found at the junction point. Two of them presented inv dup del rearrangements with a spacer between the duplications and the presence of microhomology, being proposed the Fold-back mechanism for their formation. Another patient had part of his complex rearrangement characterized despite a discrepancy between the arrays due to the presence of Low Copy Repeats (LCRs). In both patients with ring chromosomes, it was revealed the presence of a duplication followed by a terminal deletion. Conclusions: The execution of different techniques allowed for a detailed analysis of the patients' different rearrangements, which enabled the inference of its possible mechanisms of formation and the correlation of the chromosomal imbalances with the patients' phenotypes.
Background: Structural chromosomal alterations derive from breaks and rearrangements in the chromosomes and can alter the amount of genetic material present in the cell. Intrachromosomal rearrangements involve only one chromosome and include inversions, ring chromosomes and intra-arm rearrangements, such as intra-arm insertions and inv dup del rearrangements, which are characterized by an inverted duplication associated with a terminal deletion. Various mechanisms have been described to explain their formation, such as Non-Allelic Homologous Recombination (NAHR) and Non-Homologous End-Joining (NHEJ). The analysis of the DNA sequence in the breakpoints of the chromosomal alterations can reveal their mechanisms of formation as well as point out risk factors as it can expose characteristics related to DNA repair mechanisms that shape the chromosomal. rearrangements. Objective: To study rare chromosomal rearrangements involving intrachromosomal duplications and deletions. Methods: Six patients with intrachromosomal duplications and deletions were investigated by karyotyping, catalog and custom genomic array, fluorescent in situ hybridization (FISH), whole genome sequencing, and Sanger sequencing. Results: Three patients had their rearrangements completely characterized and their breakpoints sequenced. One of them presented a more complex rearrangement than expected formed by a replication mechanism which leads to the insertion of a fragment more distal to one of the breakpoints, which was supposed to be deleted but was found at the junction point. Two of them presented inv dup del rearrangements with a spacer between the duplications and the presence of microhomology, being proposed the Fold-back mechanism for their formation. Another patient had part of his complex rearrangement characterized despite a discrepancy between the arrays due to the presence of Low Copy Repeats (LCRs). In both patients with ring chromosomes, it was revealed the presence of a duplication followed by a terminal deletion. Conclusions: The execution of different techniques allowed for a detailed analysis of the patients' different rearrangements, which enabled the inference of its possible mechanisms of formation and the correlation of the chromosomal imbalances with the patients' phenotypes.