Chronic myeloid leukemia (CML) is a myeloproliferative disorder cytogenetically characterized by a reciprocal translocation between the long arms of chromosome 9 and 22 t(9;22) that leads to the formation of the BCR-ABL1 fusion gene, coding for a deregulated tyrosine kinase with oncogenic activity. In clinical routine, mRNA amount of the chimeric transcript is considered proportional to the leukemic clone and is used for the molecular monitoring of patients. However, qRT-PCR cannot identify transcriptionally silent leukemic cells that can be present in minimal residual disease (MRD). To monitor MRD it is necessary to develop a qPCR assay on DNA sequences spanning BCR-ABL1, that are patient specific. Previous results obtained by Prof. G. Porta’s group (unpublished) have demonstrated that DNA detection is positive while mRNA is not in 30% of time points, indicating the presence of transcriptionally silent cells. Breakpoints in these patients were characterized by laborious long-range PCR and cloning not suitable for a clinical application. To overcome this limiting step we set up a DNA capturing assay to target all kind of breakpoints that give rise to different BCR-ABL1 transcripts. Captured regions were then sequenced with a next generation protocol. The idea was to use the identified patient specific breakpoints to setup qPCR assays to monitor MRD. We successfully identified BCR-ABL1 fusion points in 9 over 10 samples, with single nucleotide accuracy, by setting up a bioinformatics workflow specifically developed for this purpose. All findings were validated with Sanger sequencing. This project was performed in collaboration with Prof. Giovanni Porta of University of Insubria.
MAPPING BCR-ABL1 FUSION POINTS IN CHRONIC MYELOID LEUKEMIA BY NEXT GENERATION SEQUENCING / M. Barcella ; tutore: C. Barlassina ; direttore del dottorato: M. Clerici. DIPARTIMENTO DI SCIENZE DELLA SALUTE, 2014 Feb 04. 26. ciclo, Anno Accademico 2013. [10.13130/barcella-matteo_phd2014-02-04].
MAPPING BCR-ABL1 FUSION POINTS IN CHRONIC MYELOID LEUKEMIA BY NEXT GENERATION SEQUENCING
M. Barcella
2014
Abstract
Chronic myeloid leukemia (CML) is a myeloproliferative disorder cytogenetically characterized by a reciprocal translocation between the long arms of chromosome 9 and 22 t(9;22) that leads to the formation of the BCR-ABL1 fusion gene, coding for a deregulated tyrosine kinase with oncogenic activity. In clinical routine, mRNA amount of the chimeric transcript is considered proportional to the leukemic clone and is used for the molecular monitoring of patients. However, qRT-PCR cannot identify transcriptionally silent leukemic cells that can be present in minimal residual disease (MRD). To monitor MRD it is necessary to develop a qPCR assay on DNA sequences spanning BCR-ABL1, that are patient specific. Previous results obtained by Prof. G. Porta’s group (unpublished) have demonstrated that DNA detection is positive while mRNA is not in 30% of time points, indicating the presence of transcriptionally silent cells. Breakpoints in these patients were characterized by laborious long-range PCR and cloning not suitable for a clinical application. To overcome this limiting step we set up a DNA capturing assay to target all kind of breakpoints that give rise to different BCR-ABL1 transcripts. Captured regions were then sequenced with a next generation protocol. The idea was to use the identified patient specific breakpoints to setup qPCR assays to monitor MRD. We successfully identified BCR-ABL1 fusion points in 9 over 10 samples, with single nucleotide accuracy, by setting up a bioinformatics workflow specifically developed for this purpose. All findings were validated with Sanger sequencing. This project was performed in collaboration with Prof. Giovanni Porta of University of Insubria.File | Dimensione | Formato | |
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