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This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ Optimising the use of Azathioprine in the treatment of Inflammatory Bowel Disease Smith, Melissa Ann Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). 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Sep. 2021 Optimising the use of Azathioprine in the treatment of Inflammatory Bowel Disease Dr Melissa Ann Smith MD (Res) 2013 MD thesis Azathioprine in IBD Melissa Ann Smith Table of Contents Table of Contents ............................................................................................................................ 2 Table of figures ................................................................................................................................ 5 List of Tables ................................................................................................................................... 5 Acknowledgements ......................................................................................................................... 6 Communications arising from this research .................................................................................... 7 Original Research Papers............................................................................................................. 7 Oral Presentations: ....................................................................................................................... 7 Poster Presentations ..................................................................................................................... 8 Review articles .......................................................................................................................... 10 Book Chapters ........................................................................................................................... 10 Abstract .......................................................................................................................................... 11 Chapter One: Literature Overview ............................................................................................... 12 1.1 Brief introduction to Inflammatory Bowel Disease (IBD): ................................................. 12 1.2 Thiopurine Drugs ................................................................................................................. 13 1.3 What is the evidence that thiopurines work in IBD? .......................................................... 16 1.4 How do thiopurines work? ................................................................................................. 16 1.5 Challenges to the successful use of thiopurines ................................................................. 18 1.6 What is known about thiopurine metabolism? ................................................................... 21 1.7 TPMT ................................................................................................................................. 22 1.7.1 The relationship between TPMT genotype and phenotype ......................................... 25 1.8 MTHFR .............................................................................................................................. 27 1.9 TYMS ................................................................................................................................. 30 1.10 ITPase ............................................................................................................................... 31 1.10.1 Human ITPase deficiency .......................................................................................... 33 1.10.2 Pharmacogenetic importance of ITPase deficiency .................................................. 34 1.10.3 Testing for ITPase deficiency .................................................................................... 38 1.11 Other enzymes .................................................................................................................. 39 1.11.1 Xanthine Oxidase/Dehydrogenase ........................................................................... 39 1.11.1.1 Interaction between XDH and TPMT .................................................................... 40 1.11.2 Molybdenum cofactor sulfurase ................................................................................ 41 1.11.3 Aldehyde Oxidase...................................................................................................... 42 1.11.4 Hypoxanthine Guanine Phosphoribosyltransferase (HGPRT) .................................. 42 1.11.5 Inosine monophosphate dehydrogenase (IMPDH)..................................................... 43 1.11.6 5’-nucleotidase......................................................................................................... 44 1.11.7 Nucleoside phosphokinase ....................................................................................... 44 1.11.8 MRP4 & MRP5 ........................................................................................................ 47 1.11.9 Glutathione S-transferase .......................................................................................... 54 1.11.10 Soluble –HLA-G...................................................................................................... 54 1.12 Thiopurine Metabolite Monitoring ................................................................................... 55 1.12.1 Problems with the current systems of TGN monitoring ............................................ 56 1.12.2 Alternatives to TGNs ................................................................................................ 58 Aims of the thesis: ......................................................................................................................... 59 Chapter Two: Materials and Methods ......................................................................................... 60 2.1 Patient Recruitment ............................................................................................................ 60 2 MD thesis Azathioprine in IBD Melissa Ann Smith 2.1.1 Genotyping studies (chapters 3 & 4): ........................................................................ 60 2.1.2 AOX sequencing (chapter 3) ....................................................................................... 60 2.1.3 TGN study (chapter 6) ................................................................................................ 60 2.1.4 Azathioprine and allopurinol co-treatment ................................................................... 61 2.1.4.1 Eligibility for combination therapy with azathioprine and allopurinol ..................... 61 2.2 Determination of response .................................................................................................. 62 2.2.1 Genotyping studies (chapters 3 & 4) ........................................................................... 62 2.2.2 TGN study (chapter 6) ................................................................................................. 62 2.2.3 Azathioprine / allopurinol co-treatment ..................................................................... 63 2.3 Genotyping Studies: laboratory methods .......................................................................... 64 2.3.1. SNP selection ............................................................................................................... 64 2.3.2 DNA extraction: ........................................................................................................... 65 2.3.3 Real-time PCR genotyping ......................................................................................... 66 2.3.4 Statistical Analysis ..................................................................................................... 68 2.4 Sequencing the Aldehyde Oxidase Gene in non-responders to azathioprine ..................... 68 2.4.1 Primers: ......................................................................................................................... 68 2.4.2 PCR ............................................................................................................................... 70 2.4.3 PCR Product Purification: .........................................................................................