A Study of Genomic Instability in Chronic Lymphocytic Leukaemia (CLL)



Tayeb, FJ
(2018) A Study of Genomic Instability in Chronic Lymphocytic Leukaemia (CLL). PhD thesis, University of Liverpool.

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Abstract

Chronic Lymphocytic Leukaemia (CLL) is a malignancy of mature B cells. The median age at diagnosis is 70-years old, mostly seen in Western Societies. Half of the patients show an indolent phenotype and watchful waiting is the recommended approach for their management. However, once treated, patients are heterogeneous in terms of response and relapse. Regarding prognosis, genetic testing, alongside current clinical staging, is important to guide treatment and prognosis. Deletion of the short arm of chromosome 17 (17p) is one of the worst prognostic markers for CLL and usually involves loss of heterozygosity (LOH) and mutations in the TP53 gene. P53 is one of the cell-cycle regulators that activates senescence, cell-cycle arrest, DNA repair or apoptosis as part of the DNA damage response (DDR). In some severe cases of CLL with inactivated P53, the DDR is affected in favour of CLL survival. However, there is a little knowledge regarding the relationship between TP53 and mutations affecting other DNA repair genes and whether these could lead to a synergistic effect. It was, therefore, the aim of this study to address this important question. Genomic DNA was extracted from blood CLL cells of 10 patients, all of whom were bearing mutation(s) in TP53 as identified with Sanger sequencing and had progressive disease at the time of sampling. 194 known human DNA maintenance genes were identified and biotinylated-cRNA probes designed (Agilent SureSelect) to enrich DNA from their exons (2786 regions - total size = 500kb) for sequencing using an Ion Torrent Personal Genome Machine (PGM). In terms of coverage, about 99.92% of targeted regions were successfully enriched with 297x average coverage depth. Using the Torrent 2 Variant Caller (TVC), 365 candidate missense variants in 113 genes were identified from the samples. 268 were single nucleotide variants (SNVs), and 97 were previously unknown or novel (0.002 variants per 1kbp per patient). 90% sensitivity was achieved whereas 60% specificity resulted from a high rate of false positives (FP) found as homopolymer indels. Each of two out of the 10 samples (20%) had separate POLE novel missense mutations, which were validated by Sanger and Whole Genome Sequencing (WGS). This was further investigated with an expanded cohort of patients divided according to TP53 status into TP53 wild-type (n=28) and TP53 mutated (n=31). The results showed no further POLE mutation in the cohort (3.39%) and confirmed the independent role of TP53 pathogenesis in CLL. Whole-genome Sequencing (WGS) to a lesser depth was also applied to the same primary cohort of ten CLL samples. Coverage analysis demonstrated there to be a 98.5% average base coverage and 29.7x average coverage depth. Data analysis found an average of 250 novel missense variants (2.5x10-5 variants per 1kbp per sample). The data also confirmed the 17p deletions and mutations. Genotyping data shows that many genes could be affected, involving signal transduction and immune response pathways that may participate in B cell development and CLL pathogenesis affected novel cells, supporting the possibility that oncogenes may initiate CLL carcinogenesis prior to TP53 mutation and chromosomal instability. Taken together, these results show that there are mutations in DNA repair genes but they are not common, at least for the samples examined. This suggests the independent role of P53 in deactivating DNA repairing mechanisms. Further validation should be applied 3 using a larger cohort. Furthermore, NGS proved to be a comprehensive tool for examining a group of genes or even genomes in a robust manner for characterising CLL.

Item Type: Thesis (PhD)
Additional Information: My Permanent address: 7684 Alfakhr Alfarsi St, Alnuzha District, Jeddah, 3968-23534 My correspondent address: 103 Benson St, Liverpool, L1 2SS, the UK
Divisions: Faculty of Health and Life Sciences > Faculty of Health and Life Sciences
Depositing User: Symplectic Admin
Date Deposited: 23 Aug 2018 14:00
Last Modified: 08 Apr 2021 11:05
DOI: 10.17638/03019754
Supervisors:
  • Pettitt, Andrew
  • Sibson, David Ross
  • Lin, Ke
URI: https://livrepository.liverpool.ac.uk/id/eprint/3019754