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Title: RNA-sequencing for investigation of gene fusions and splicing abnormalities in leukaemia
Author: Knut, Marcin
ISNI:       0000 0004 6348 7004
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2016
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Myeloid malignancies, a group of neoplasms affecting myeloid blood lineages, are driven by a variety of somatically acquired mutations and gene fusions. Some of these genes, e.g. U2AF1, encode factors involved in pre-mRNA splicing but it remains unclear how these abnormalities contribute to malignancy. The presence of gene fusions is typically signalled by the finding of a somatic chromosome translocation but it is unclear to what extent leukaemia might be driven by cryptic abnormalities that have escaped detection by cytogenetic analysis. This study focuses on the use of RNA-Seq by Next Generation Sequencing (NGS) to detect gene fusions and help understand the role of U2AF1. Two bioinformatic pipelines were developed. The fusion detection pipeline combined with manual selection of candidates achieved 100% accuracy across 5 samples with previously discovered, cytogenetically visible gene fusions. Across 4 samples, for which gene fusions were suspected, the developed methods allowed for identification and validation of at least 1 gene fusion in each sample, and up to 5 within a single sample, with overall accuracy of 29%. Discovery of BCR-JAK2 gene fusion in one of the samples explained patient's short-termed remission following application of JAK1/JAK2 inhibitor ruxolitinib which highlights importance of RNA-Seq as a tool potentially leading to targeted therapy. A knock- down was performed on U2AF1 and both of its isoforms in a cell line. The analysis using second bioinformatic pipeline showed that U2AF1 depletion modified RNA processing events, disproportion- ally affecting usage of terminal exons over others. Observed upregulated exons in depleted cells had longer AG exclusion zones (AGEZ) and polypyrimidine tracts (PPTs). A small number of transcripts responded differently to knockdowns of specific isoforms. These results provide new insights into gene fusions as well as U2AF1 function to better understand myeloid malignancies.
Supervisor: Cross, Nicholas ; Collins, Andrew ; Ennis, Sarah ; Tapper, William Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available