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Title: Investigation of the pharmacogenetics of colorectal cancer
Author: Coffey, Michelle
ISNI:       0000 0004 5917 8372
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2016
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In this thesis, we aimed to elucidate the functional effects of three rare nonsynonymous variants in ERCC4 on cell viability, DNA repair and localisation of the ERCC1-XPF repair complex. We also aimed to identify alleles that contribute to extreme adverse reactions to colorectal cancer (CRC) treatment. Previous work identified three rare nonsynonymous variants (Pro379Ser, Arg576Thr and Glu875Gly) in the ERCC4 gene of patients with severe peripheral neuropathy associated with oxaliplatin (PNAO). All variants were predicted to affect protein function, whilst two collectively contributed to the risk of the phenotype (11.11% of patients with PNAO vs. 4.88% of patients without PNAO; P = 0.03). We genotyped 480 EBV-transformed lymphoblastoid cell lines from healthy humans for ERCC1 and ERCC4 polymorphisms. From this data, we selected twelve cell lines for functional analysis (three wild type and three cell lines per ERCC4 variant). Following treatment with UV or oxaliplatin, we observed negligible differences in survival between wild type and variant cell lines. We identified negligible differences in localisation of the ERCC1-XPF repair complex between wild type and variant cell lines after oxaliplatin treatment. Using the haploid fission yeast, Schizosaccharomyces pombe, similarly we observed minimal differences in rad16 localisation between wild type and variant strains. We observed similar repair rates following oxaliplatin treatment in wild type and variant cell lines. However, following UV treatment, all cell lines carrying Pro379Ser exhibited retarded repair (defined as virtually no repair for the duration of the experiment) whilst all cell lines carrying Arg576Thr and Glu875Gly displayed delayed repair commencing 24 hours after damage. This suggested a functional defect of XPF in the repair of UV damage in cells carrying an ERCC4 variant. We used whole exome resequencing data from 10 patients with PNAO and 45 patients without PNAO, to identify causal alleles for the phenotype. We validated one variant in PAPLN, suggesting that this gene may cause PNAO. We identified a high rate of false-positive indel calls (98.1%), of which 96.1% contained homopolymers or short tandem repeats (STRs) in their flanking regions. Additionally, we used five filtering strategies to identify causal variants and manually excluded indels that were likely false-positives. In addition to PAPLN, this identified a further seven genes (ASGR2, ANXA7, OR2T35, SLC17A5, SMOC2, SPERT and TAF1D) that may cause PNAO.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: RC0254 Neoplasms. Tumors. Oncology (including Cancer)