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Investigating the Genetic Basis of Cisplatin-Induced Ototoxicity in Adult South African Patients

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Investigating the Genetic Basis of Cisplatin-Induced Ototoxicity in Adult South African Patients --------------------------------------------------------------------------- Investigating the genetic basis of cisplatin-induced ototoxicity in adult South African patients --------------------------------------------------------------------------- by Timothy Francis Spracklen SPRTIM002 SUBMITTED TO THE UNIVERSITY OF CAPE TOWN In fulfilment of the requirements for the degree MSc(Med) Faculty of Health Sciences UNIVERSITY OF CAPE TOWN University18 December of Cape 2015 Town Supervisor: Prof. Rajkumar S Ramesar Co-supervisor: Ms A Alvera Vorster Division of Human Genetics, Department of Pathology, University of Cape Town 1 The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town Declaration I, Timothy Spracklen, hereby declare that the work on which this dissertation/thesis is based is my original work (except where acknowledgements indicate otherwise) and that neither the whole work nor any part of it has been, is being, or is to be submitted for another degree in this or any other university. I empower the university to reproduce for the purpose of research either the whole or any portion of the contents in any manner whatsoever. Signature: Date: 18 December 2015 ' 2 Contents Abbreviations ………………………………………………………………………………….. 1 List of figures …………………………………………………………………………………... 6 List of tables ………………………………………………………………………………….... 7 Abstract ………………………………………………………………………………………… 10 1. Introduction …………………………………………………………………………………. 11 1.1 Cancer …………………………………………………………………………….. 11 1.2 Adverse drug reactions ………………………………………………………….. 12 1.3 Cisplatin …………………………………………………………………………… 12 1.3.1 Cisplatin’s mechanism of action ……………………………………………… 13 1.3.2 Adverse reactions to cisplatin therapy ………………………………………. 14 1.3.2.1 Cisplatin-induced ototoxicity ………………………………………………... 14 1.3.2.2 Mechanism of cisplatin-induced ototoxicity ………………………………. 16 1.4 Audiometry ………………………………………………………………………. 19 1.5 Risk factors of ototoxicity ………………………………………………………. 20 1.6 Pharmacogenetics ………………………………………………………………. 21 1.6.1 The pharmacogenetics of cisplatin-induced ototoxicity ……………………. 21 1.6.1.1 Genes involved in drug metabolism and detoxification ………………….. 23 1.6.1.1.1 The glutathione S-transferases …………………………………………. 23 1.6.1.1.2 Methyltransferases ……………………………………………………….. 24 1.6.1.1.3 Dehydrogenases …………………………………………………………... 25 1.6.1.2 Membrane receptors and transporters ……………………………………. 25 1.6.1.3 Deafness-associated genes ……………………………………………….. 26 1.6.1.4 Other genes ………………………………………………………………….. 27 1.7 Future considerations or directions …………………………………………….. 28 1.8 Research aim and objectives …………………………………………………… 29 2. Analysis of clinical and demographic risk factors of cisplatin-induced ototoxicity …... 30 2.1 Introduction ……………………………………………………………………….. 30 2.2 Methods …………………………………………………………………………… 31 2.2.1 Patients …………………………………………………………………………. 31 2.2.2 Treatment details ………………………………………………………………. 32 2.2.3 Audiometric monitoring ………………………………………………………... 32 2.2.4 Statistical analyses …………………………………………………………….. 33 2.3 Results …………………………………………………………………………….. 33 3 2.3.1 Patient cohort …………………………………………………………………... 33 2.3.2 Association of clinical characteristics with ototoxicity ……………………… 34 2.3.3 Association of demographic variables with ototoxicity …………………….. 35 2.3.4 Statistical modelling of cisplatin-induced ototoxicity ……………………….. 37 2.4 Discussion ………………………………………………………………………… 38 2.5 Conclusion ………………………………………………………………………… 40 3. Investigation of candidate variants and their role in cisplatin-induced ototoxicity …… 42 3.1 Introduction ……………………………………………………………………….. 42 3.2 Methods …………………………………………………………………………… 45 3.2.1 Research design ……………………………………………………………….. 45 3.2.2 Patient recruitment …………………………………………………………….. 45 3.2.2.1 Inclusion and exclusion criteria …………………………………………….. 45 3.2.2.2 Collection of data, biological samples, and informed consent ………….. 46 3.2.2.3 Audiometric analysis ………………………………………………………… 46 3.2.3 DNA extraction and quality control ………………………………………….. 47 3.2.4 Candidate selection and the amplification of target DNA regions ………... 47 3.2.4.1 PCR conditions ………………………………………………………………. 48 3.2.4.2 Purification of PCR products ………………………………………………. 49 3.2.5 SNaPshot® genotyping ……………………………………………………….. 49 3.2.5.1 Visualisation and analysis of SNaPshot® reaction products ……………. 50 3.2.6 Validation of genotyping ………………………………………………………. 50 3.2.6.1 Direct cycle sequencing reaction conditions ……………………………… 51 3.2.6.2 Analysis of sequencing products …………………………………………... 51 3.2.7 Statistical analysis of data …………………………………………………….. 52 3.3 Results …………………………………………………………………………….. 53 3.3.1 Patient cohort …………………………………………………………………... 53 3.3.2 Incidence of ototoxicity in the cohort ………………………………………… 54 3.3.3 Candidate gene study …………………………………………………………. 56 3.3.3.1 Single site analysis ………………………………………………………….. 56 3.3.3.2 Haplotype analysis …………………………………………………………... 61 3.3.3.3 Regression modelling of clinical, demographic and genetic variables ……………………………………………………………………………….. 62 3.3.3.4 Other markers of treatment response ……………………………………... 64 3.4 Discussion ………………………………………………………………………… 65 3.5 Conclusion ………………………………………………………………………… 70 4. Whole-exome sequencing to identify potential modifiers of drug response …………. 71 4.1 Introduction ……………………………………………………………………….. 71 4 4.2 Methods …………………………………………………………………………… 73 4.2.1 Patients and samples ………………………………………………………… 73 4.2.2 WES ……………………………………………………………………………. 73 4.2.3 Variant annotation ……………………………………………………………... 74 4.2.4 Scoring and filtering of exonic variants ……………………………………… 75 4.2.4.1 Variant-level analysis ……………………………………………………….. 75 4.2.4.1.1 Analysis of gene-disease relationships …………………………………. 75 4.2.4.1.2 Gene prioritisation …………………………………………………………. 76 4.2.4.2 Gene-level analysis …………………………………………………………. 76 4.2.4.2.1 Pathway analysis …………………………………………………………. 76 4.2.4.2.2 Panel analysis ……………………………………………………………... 77 4.2.5 Statistical analysis of patient demographic and genomic data …………… 77 4.3 Results …………………………………………………………………………….. 78 4.3.1 Extreme-phenotype cohort ……………………………………………………. 78 4.3.2 WES ……………………………………………………………………………... 83 4.3.2.1 Principal component analysis ………………………………………………. 86 4.3.2.2 Filtering of variants …………………………………………………………. 88 4.3.2.2.1 Variant-based filtering …………………………………………………….. 91 4.3.2.2.2 Gene-based filtering ………………………………………………………. 92 4.3.2.2.2.1 Panel-based gene-level analysis ……………………………………… 96 4.3.2.2.2.2 Pathway gene-level analysis ………………………………………….. 98 4.4 Discussion ………………………………………………………………………… 101 4.5 Conclusion ………………………………………………………………………… 111 5. Conclusion and future perspectives ……………………………………………………… 112 6. References ………………………………………………………………………………….. 114 6.1 Literature cited …………………………………………………………………… 114 6.2 Websites accessed ………………………………………………………………. 133 Appendices …………………………………………………………………………………….. 136 Appendix A: Letter of ethical approval …………………………………………….. 136 Appendix B: Molecular study information sheet provided to patients during recruitment …………………………………………………………………….. 137 Appendix C: Informed consent form for access to patient records ……………… 139 Appendix D: Informed consent for the collection, analysis and storage of patient biological samples ………………………………………………. 140 Appendix E: Letter of GSH approval ……………………………………………….. 143 Appendix F: Analysis of threshold changes in all prospective and retrospective patients ………………………………………………………………… 144 5 Appendix G: DNA extraction protocols …………………………………………….. 146 Appendix H: Buffers and reagents ………………………………………………….. 148 Appendix I: Tests of association and regression between genotypic and allelic information and ototoxicity in the total patient cohort ………………… 150 Appendix J: Gene panels used in analysis of WES data ………………………… 154 Appendix K: Gene-disease relationships for WES data ………………………….. 165 Appendix L: Panel-based analysis of all unique variants scoring ≥ 4 …………... 169 Appendix M: Results of pathway analysis …………………………………………. 174 Appendix N: Pathway panels ……………………………………………………….. 176 Acknowledgements …………………………………………………………………………… 193 Turnitin: cover page …………………………………………………………………………… 194 Turnitin: summary of originality report ………………………………………………………. 195 6 Abbreviations 5-FU: 5-fluorouracil A: adenine ABC: ATP-binding cassette ACYP2: acylphosphatase 2, muscle type ADA: adenosine deaminase ADME: drug absorption, distribution, metabolism and elimination ADR: adverse drug reaction AFR: 1000 Genomes Project African population AMPK: AMP-activated protein kinase AMR: 1000 Genomes Project admixed American population AP: alkaline phosphatase AQP: aquaporin ARE: antioxidant response element Arntl: aryl hydrocarbon receptor nuclear translocator-like ASHA: American Speech-Language-Hearing Association ASN: 1000 Genomes Project East Asian population ATP: adenosine triphosphate BA: black African BCL2: B-cell CLL/lymphoma 2 bp: base pairs C: cytosine C2: complement component 2 C3: complement component 3 C5: complement component 5 CAF: Central Analytical Facility cAMP: cyclic adenosine monophosphate CAT: catalase Cau: Caucasian CCND1: cyclin D1 CDH13: cadherin 13 CFB: complement factor B CFTR: cystic fibrosis transmembrane conductance regulator CI: confidence interval 1 COCH: cochlin COMT: catechol-O-methyltransferase
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