Implementação do diagnóstico de dissomia uniparental para os cromossomas humanos 7, 11, 13, 14, 15, 21 e 22 no Laboratório de Citogenética e Genómica da Faculdade de Medicina da Universidade de Coimbra
Ficheiros
Data
2013
Autores
Título da revista
ISSN da revista
Título do Volume
Editora
Resumo
A dissomia uniparental (UPD - uniparental disomy) define-se pela presença de um par de cromossomas numa linha celular dissómica proveniente apenas de um único progenitor. Esta pode surgir para o complemento cromossómico total, para um par de cromossomas completo, ou apenas num segmento cromossómico juntamente com a herança biparental do resto deste par de cromossomas.
O objetivo deste trabalho foi implementar e validar o diagnóstico da UPD para os cromossomas 7, 11, 13, 14, 15, 21, 22. A fim de cumprir este objetivo estabeleceu-se a análise da herança alélica através da amplificação simultânea de vários loci de STRs (short tandem repeats) ao longo do cromossoma de interesse por multiplex PCR (polymerase chain reaction), seguido da separação dos fragmentos marcados por eletroforese capilar. Além da análise por STRs, estudos de metilação e do número de cópias das regiões 11p15 e 15q11, sujeitas a imprinting, foram efetuados através da técnica de MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification). Os métodos baseados em metilação permitem detetar as alterações moleculares que modificam o padrão de metilação biparental em uniparental: UPD, microdeleções, epimutações e mutações no ICR (imprinting control region)/ DMR (differentially methylated region).
A análise da herança alélica de casos clínicos com aneuploidias permitiu concluir que os primers de cada multiplex PCR amplificam com especificidade os STRs pretendidos. A análise da herança alélica para despiste de UPD dos cromossomas 11, 14, 15 e 21, foi validada através de casos clínicos estudados previamente por outro laboratório. No entanto, a análise da herança alélica no diagnóstico de UPD revelou que a quantidade de STRs estudada em cada multiplex PCR foi reduzida, especialmente para as regiões contendo genes sujeitos a imprinting. Sugerindo portanto que sejam desenhados mais primers para amplificar STRs dentro de regiões sujeitas a imprinting, para introduzir ao respetivo multiplex PCR.
Uniparental disomy (UPD) is defined by the presence of a chromosome pair derived only from one parent present in a disomic cell line. It can occur for the entire chromosomal complement, for a complete chromosome or only in a chromosomal segment together with biparental inheritance of the rest of the pair of the chromosome. The aim of this work was to implement and validate the diagnosis of UPD chromosomes 7, 11, 13, 14, 15, 21, 22. In order to achieve this goal the analysis of allelic inheritance was established by amplification of multiple loci STRs (short tandem repeat) along the chromosome of interest by multiplex PCR (polymerase chain reaction), followed by separation of the labeled fragments by capillary electrophoresis. In addition to the analysis of STRs studies of copy number and methylation of regions 11p15 and 15q11, subject to imprinting, were made through the technique of MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification). The methylation-based methods can detect molecular defects that turn a biparental into a uniparental methylation pattern: UPD, microdeletions, epimutations, ICR (imprinting control region)/ DMR (differentially methylated region) mutations. The analysis of allelic inheritance of clinical cases with aneuploidy allowed concluding that the primers of each multiplex PCR specifically amplify the STRs intended. The analysis of allelic inheritance in order to screening UPD of the chromosomes 11, 14, 15 and 21 was validated through clinical cases studied previously by another laboratory. However, the analysis of allelic inheritance in the diagnosis of UPD revealed that the number of STRs studied in each multiplex PCR is reduced, especially for the regions containing genes subject to imprinting, suggesting therefore that more primers are drawn to amplify STRs, which are present in regions subject to imprinting, to introduce to the corresponding multiplex PCR.
Uniparental disomy (UPD) is defined by the presence of a chromosome pair derived only from one parent present in a disomic cell line. It can occur for the entire chromosomal complement, for a complete chromosome or only in a chromosomal segment together with biparental inheritance of the rest of the pair of the chromosome. The aim of this work was to implement and validate the diagnosis of UPD chromosomes 7, 11, 13, 14, 15, 21, 22. In order to achieve this goal the analysis of allelic inheritance was established by amplification of multiple loci STRs (short tandem repeat) along the chromosome of interest by multiplex PCR (polymerase chain reaction), followed by separation of the labeled fragments by capillary electrophoresis. In addition to the analysis of STRs studies of copy number and methylation of regions 11p15 and 15q11, subject to imprinting, were made through the technique of MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification). The methylation-based methods can detect molecular defects that turn a biparental into a uniparental methylation pattern: UPD, microdeletions, epimutations, ICR (imprinting control region)/ DMR (differentially methylated region) mutations. The analysis of allelic inheritance of clinical cases with aneuploidy allowed concluding that the primers of each multiplex PCR specifically amplify the STRs intended. The analysis of allelic inheritance in order to screening UPD of the chromosomes 11, 14, 15 and 21 was validated through clinical cases studied previously by another laboratory. However, the analysis of allelic inheritance in the diagnosis of UPD revealed that the number of STRs studied in each multiplex PCR is reduced, especially for the regions containing genes subject to imprinting, suggesting therefore that more primers are drawn to amplify STRs, which are present in regions subject to imprinting, to introduce to the corresponding multiplex PCR.
Descrição
Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde
Palavras-chave
Dissomia uniparental , Cromossomas