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Investigation of the Pharmacogenetics of Colorectal Cancer

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Investigation of the Pharmacogenetics of Colorectal Cancer Investigation of the Pharmacogenetics of Colorectal Cancer Submitted for the degree of Doctor of Philosophy at Cardiff University Michelle Coffey 2015 DECLARATION This work has not previously been accepted in substance for any degree and is not concurrently submitted in candidature for any degree. Signed - Date – STATEMENT 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of PhD Signed - Date – STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. Signed - Date – STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed ………………………………………… (candidate) Date ……………… STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loans after expiry of a bar on access previously approved by the Graduate Development Committee. Signed - Date - ii Summary 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. iii Acknowledgments I would like to thank the following: My supervisors, Prof. Jeremy Cheadle and Dr. Richard Adams, for their supervision and guidance throughout this project. Tenovus for funding this work. Chris George for his extensive help with the immunofluorescence assay, in terms of training on the confocal microscope and assistance with troubleshooting. Catherine Naseriyan for help with FACS and technical advice. Oliver Fleck for his contribution to Schizosaccharomyces pombe strain construction and phenotype testing, as well as help with culturing and freezing down strains. David Fisher for providing information about the patients with severe peripheral neuropathy for the sequencing study. All of the patients in the MRC COIN trial who generously donated DNA, without whom the project would not have been possible. Simon Reed and Richard Webster for their advice. Special thanks to Hannah West for her advice and help with various techniques in the lab, guidance for writing up and for all the background information for this project. Thanks to Chris Smith and James Colley for help and advice with writing up. Special thanks to Marc Naven for help with bioinformatic analysis and information during writing up. Shelley for help and advice regarding technical issues. Laura Thomas for advice about cell culture and for providing a kind ear when needed. Pete and Julie for technical help and providing a nice atmosphere in the lab. Rebecca Harris for help with sequencing in the early days. The administration team, Linda, Sherrie, Sathiya, Sally, Alma, Beth and Mark, for helping out with technical things. Dave Millar and Andy Tee for advice about cell culture. Marc, Matt, Laura, Ellie, Sara, Kayleigh, Elaine and my friends back home for making the PhD enjoyable. Carl, for his love and continuous support throughout this process. Mum, Dad, Elaine and Paul, for their love and support. iv Abbreviations ABC ATP-binding cassette aCRC Advanced colorectal cancer ADCC Antibody-dependent cell-mediated cytotoxicity ADME Absorption, distribution, metabolism and excretion AGXT Alanine glycoxylate transferase Align-GVGD Align-Grantham Variation/Grantham Deviation AOA1 Ataxia with oculomotor apraxia 1 APTX Aprataxin AT Ataxia telangiectasia ATM Ataxia telangiectasia, mutated ATP Adenosine triphosphate AU Arbitrary units BER Base excision repair BGI Beijing Genomics Institute BHRF1 BamHI-H right reading frame-1 BLM Bloom syndrome BPDE Benzo(a)pyrene diolepoxide BRAF V-raf murine sarcoma viral oncogene homolog B1 BRCA1/2 Breast cancer 1/2, early onset BSA Bovine serum albumin BWA Burrows-Wheeler Alignment CBS Central Biotechnology Services CCNH Cyclin H CCS Copper chaperone for superoxide dismutase CEA Carcinoembryonic antigen CFTR Cystic fibrosis transmembrane conductance regulator ChIP Chromatin immunoprecipitation ChIP-chip Chromatin immunoprecipitation combined with microarray ChIP-seq Chromatin immunoprecipitation combined with sequencing CMT Charcot-Marie-Tooth disease CMT1A Charcot-Marie-Tooth disease type 1A CMT2A Charcot-Marie-Tooth disease type 2A v COIN Continuous vs. intermittent therapy Co-IP Co-immunoprecipitation COX17 Cytochrome C oxidase 17 CPB Capecitabine CPD Cyclobutane pyrimidine dimer CRA Colorectal adenoma CRC Colorectal cancer CS Cockayne syndrome CSA/B Cockayne syndrome, complementation group A/B CTR1 Copper transporter protein 1 CYP Cytochrome P DABCO 1,4-diazabicyclo[2.2.2]octane DACH 1,2-diaminocyclohexane DAPI 4’,6-diamidino-2-Phenylindole, dihydrochloride DDB1/2 DNA damage binding protein 1/2 ddNTPs Dideoxyribonucleotide triphosphates DFUR 5’-deoxy-5-fluorouridine dH2O Distilled water DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTPs Deoxyribonucleotide triphosphates DPN Diabetic peripheral neuropathy DPYD Dihydropyrimidine dehydrogenase DRG Dorsal root ganglia DSB Double strand break DSBR Double strand break repair EBNA1-3C Epstein-Barr nuclear antigen 1-3C EBNA-LP Epstein-Barr nuclear antigen-leader protein EBV Epstein-Barr virus ECACC European Collection of Cell Cultures EGFR Epidermal growth factor receptor EGTA Ethylene-bis(oxyethylenenitrilo)tetraacetic acid EL Ectopia lentis ELISA Enzyme-linked immunosorbent assay ER Oestrogen receptor vi ERCC(1-6) Excision repair cross complementation group 1-6 Exo Exonuclease FA Fanconi anaemia FACS Fluorescence activated cell sorting FAD Flavin adenine dinucleotide FAP Familial adenomatous polyposis FBS Foetal bovine serum FDA US food and drug administration fdUMP Fluorodeoxyuridine monophosphate fdUTP Fluorodeoxyuridine triphosphate FOLFIRI 5-Fluorouracil, leucovorin and irinotecan FOLFOX 5-Fluorouracil, leucovorin and oxaliplatin FSC-A Forward scatter according to area FSC-H Forward scatter according to height FUTP Fluorouridine triphosphate GATK Genome Analysis Toolkit GBM Glioblastoma multiforme GC Guanine-cytosine base pair GD Geleophysic dysplasia GG-NER Global genomic nucleotide excision repair GI Gastrointestinal GRHPR Glycoxylate reductase-hydroxypyruvate reductase GST Glutathione S-transferase GWAS Genome-wide association study HAH1 Human antioxidant homologue 1 HNPCC Hereditary nonpolyposis colorectal cancer HPA Health Protection Agency HR Homologous recombination HRP Horseradish peroxidase ICL Interstrand crosslink ICLR Interstrand crosslink repair Indel Insertion or deletion kb Kilobase kDa Kilo-Dalton KRAS Kirsten rat sarcoma viral oncogene homolog vii LD Linkage disequilibrium LDH Lactate dehydrogenase LIG4 DNA Ligase IV LM Ligation mediated LMP1-2B Latent membrane protein1-2B MAF Minor allele frequency MAP MUTYH associated polyposis Mb Megabase MCL Metachromatic leucodystrophy mCRCMetastatic colorectal cancer MCV Motor conduction velocity MFN2 Mitofusin-2 MLPR Multiplex ligation-dependent probe amplification MMC Mitomycin C MMR Mismatch repair MP Maximum projection MRC Medical Research Council mRNA Messenger ribonucleic acid MSC Mesenchymal stem cell
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