THE RELATIONSHIP BETWEEN METHYLATION AND GENES ASSOCIATED WITH OPIOID RESPONSE IN HUMANS. Poppy McLaughlin A thesis submitted in partial fulfilment of the requirements of Bournemouth University for the degree of Doctor of Philosophy April 2016 This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and due acknowledgement must always be made of the use of any material conducted in, or derived from, this thesis. II ABSTRACT “The relationship between methylation and genes associated with opioid response in humans.” Poppy McLaughlin Opioids are used to alleviate pain however ~10–30% of the Caucasian population obtain ineffective pain relief and / or intolerable side effects. Genetic polymorphisms in opioid pharmacodynamic and pharmacokinetic important genes have been investigated however there has been a lack of conclusive results. Epigenetic gene regulation is another study field of interest. DNA methylation is a gene regulatory mechanism that has been associated with gene repression or expression. Thus it was hypothesised that variable opioid response was influenced by methylation alterations. The promoter region methylation of ABCB1/MDR1, CYP2D6 and OPRM1 genes were analysed by pyrosequencing in smoking, opioid exposed and control pilot populations. Genetic variations within ABCB1/MDR1, COMT, CYP2B6, CYP2D6 and OPRM1 genes were also investigated. Tissue opioid concentrations were determined by HPLC- MS/MS and GC-MS/MS. DNA methylation was influenced by chronic opioid exposure and / or the lifestyle associated with opioid dependency, but not by cigarette smoke exposure. An increase in DNA methylation was observed in the opioid exposed baby population but this was not indicative of development of neonatal abstinence syndrome (NAS). Associations between the CYP2B6*6 genotype and NAS development were found. No relationship was observed between gene methylation and opioid response but variants in OPRM1 and ABCB1/MDR1 exhibited a relationship with opioid response in cancer pain. This investigative pilot research revealed that some genetic variants can be used as diagnostic markers to predict susceptibility of methadone exposed babies to NAS development, and others are associated with variable opioid response in a cancer patient population. Although DNA methylation was not observed to be a contributory factor to opioid response, this research has ascertained the influence of chronic opioid exposure on DNA methylation of various biological samples. This finding is significant for future studies. III TABLE OF CONTENTS Abstract III Table of Contents IV Table of Figures IX Table of Tables XII Acknowledgements XIV Authors Declaration XV List of Abbreviations XVI CHAPTER 1.0 INTRODUCTION 1 1.1 Study rationale 3 1.2 Study aim 4 1.2.1 Objectives 4 CHAPTER 2.0 LITERATURE REVIEW 5 2.1 Opioids 5 2.1.1 Opioid adverse effects 9 2.1.1.1 Opioid tolerance 10 2.1.1.2 Opioid dependence 11 2.1.2 Opioid pharmacodynamics 12 2.1.2.1 µ-Opioid receptor distribution 12 2.1.2.2 µ-Opioid receptor structure 13 2.1.2.3 µ-Opioid receptor signal transduction 14 2.1.2.4 µ-Opioid receptor desensitisation, recycling and degradation 16 2.1.2.5 µ-Opioid receptor subtypes 18 2.1.2.5.1 Receptor splice variants 20 2.1.3 Opioid pharmacokinetics 23 2.1.3.1 Absorption and bioavailability 23 2.1.3.2 Metabolism 27 2.1.3.2.1 Morphine 27 2.1.3.2.2 Oxycodone 28 2.1.3.2.3 Methadone 29 2.1.3.3 Distribution 30 2.1.3.4 Elimination 32 2.1.4 Factors that influence opioid response 32 2.1.4.1 Age and opioid response 33 2.1.4.2 Organ dysfunction 34 IV 2.1.4.3 Choice, dose and route of opioid administration 34 2.1.4.4 Drug-drug interactions 35 2.1.4.5 Prior opioid experience 36 2.1.4.6 Genetic variations 36 2.2 DNA methylation 42 2.2.1 Functions of DNA methylation 45 2.2.2 DNA methyltransferases (DNMTs) 48 2.2.2.1 De novo methylation and maintenance methylation 48 2.2.3 Mechanism of gene regulation by DNA methylation 51 2.2.4 Factors that influence DNA methylation 55 2.2.4.1 Diet 55 2.2.4.2 Age 58 2.2.4.3 Environmental and drug exposures 58 2.2.4.4 Smoking 60 2.2.5 DNA methylation and opioid genes 63 2.2.6 DNA methylation analysis 65 CHAPTER 3.0 GENERIC METHODS 69 3.1 Ethical considerations and patient recruitment 69 3.1.1 Does smoking affect buccal DNA methylation? 69 3.1.2 Methadone-prescribed opioid-dependant mothers and their new-born babies 70 3.1.3 Tissue specific DNA methylation 72 3.1.3.1 Dundee blood and tissue samples 72 3.1.3.2 Edinburgh tissue samples 72 3.1.4 Pilot trial: Personalising opioid therapy for cancer pain relief 73 3.1.4.1 Royal Bournemouth, Christchurch and Poole Hospitals 73 3.1.4.2 Royal Marsden Hospital 77 3.2 Sample collection 78 3.2.1 Does smoking affect buccal DNA methylation? 79 3.2.1.1 Oral swabs 79 3.2.2 Methadone-prescribed opioid-dependant mothers and their new-born babies 79 3.2.2.1 Oral swabs 79 3.2.2.2 Plasma samples 79 3.2.3 Tissue specific DNA methylation 80 3.2.3.1 Blood samples 80 3.2.3.2 Tissue samples 80 3.2.4 Pilot trial: Personalising opioid therapy for cancer pain relief 81 3.2.4.1 Oral swabs 81 3.2.4.2 Blood samples 81 3.3 Toxicological analysis 82 V 3.3.1 Methadone-prescribed opioid-dependant mothers and their new-born babies 82 3.3.2 Tissue specific DNA methylation 83 3.3.2.1 Determination of methadone – opioid overdoses 83 3.3.2.2 Determination of morphine – opioid overdoses 83 3.3.3 Pilot trial: Personalising opioid therapy for cancer pain relief 84 3.4 SNP and gene duplication/deletion analysis 86 3.4.1 DNA extraction 86 3.4.1.1 DNA from buccal swabs 86 3.4.1.2 DNA from blood and tissue 86 3.4.1.3 DNA from plasma 87 3.4.2 DNA quantification 87 3.4.3 DNA preparation and packaging for LGC 88 3.4.3.1 DNA quantity and volume 91 3.4.3.2 Packaging, transportation 92 3.5 DNA methylation analysis 93 3.5.1 Pyrosequencing method – trial 93 3.5.1.1 Primer design 93 3.5.1.2 Bisulfite conversion 94 3.5.1.3 PCR optimisation 95 3.5.1.4 ABI310 genetic analyser 98 3.5.1.5 Pyrosequencing 100 3.5.2 Designing methylation assays for ABCB1/MDR1 and CYP2D6 102 3.5.2.1 Primers for ABCB1/MDR1 and CYP2D6 103 3.5.2.2 Bisulfite conversion 104 3.2.2.3 PCR amplification 104 3.6 Statistical analysis 106 3.6.1 Normally distributed data 106 3.6.2 Non-normally distributed data 106 CHAPTER 4.0 DOES SMOKING INFLUENCE BUCCAL DNA METHYLATION? 107 4.1 Introduction 107 4.2 Materials and methods 107 4.2.1 Sample collection 107 4.2.2 Methylation analysis 107 4.3 Results 108 4.4 Discussion 111 4.4.1 Study limitations 112 CHAPTER 5.0 METHADONE-PRESCRIBED OPIOID-DEPENDANT MOTHERS AND THEIR NEW-BORN BABIES: Babies Born to Methadone- VI Prescribed Opioid-Dependant Mothers have Elevated DNA Methylation on ABCB1/MDR1, CYP2D6 and OPRM1 113 5.1 Introduction 113 5.2 Materials and methods 114 5.2.1 Sample collection 114 5.2.2 Toxicological analysis 114 5.2.3 DNA methylation analysis 115 5.3 Results 116 5.3.1 Methylation differences between methadone exposed and non-opioid exposed mothers and babies 116 5.3.2 Relationship between methylation and smoking / residential status 118 5.3.3 Relationship between methylation and NAS development 119 5.3.4 Methylation differences between paired plasma and buccal DNA samples 120 5.4 Discussion 121 5.4.1 Study limitations 126 CHAPTER 6.0 METHADONE-PRESCRIBED OPIOID-DEPENDANT MOTHERS AND THEIR NEW-BORN BABIES: The CYP2B6*6 polymorphism protects babies exposed to methadone in utero from neonatal abstinence syndrome development 127 6.1 Introduction 127 6.2 Materials and methods 129 6.2.1 Sample collection 129 6.2.2 Toxicological analysis 129 6.2.3 SNP and gene duplication / deletion analysis 129 6.3 Results 130 6.3.1 Associations between ABCB1/MDR1, COMT, CYP2B6, CYP2D6 and OPRM1 genes and NAS development 131 6.3.2 Genetic variations and methadone dose and plasma concentrations 134 6.4 Discussion 136 6.4.1 Study limitations 137 CHAPTER 7.0 TISSUE SPECIFIC DNA METHYLATION: DNA methylation of ABCB1/MDR1, CYP2D6 and OPRM1 in individuals whose deaths were attributed to heroin toxicity 139 7.1 Introduction 139 7.2 Materials and methods 141 7.2.1 Sample collection 141 7.2.2 Toxicological analysis 141 VII 7.2.3 SNP and gene duplication / deletion analysis 141 7.2.4 DNA methylation analysis 141 7.3 Results 142 7.3.1 Age and methylation 143 7.3.1.1 ABCB1/MDR1 DNA methylation and age 143 7.3.1.2 CYP2D6 DNA methylation and age 144 7.3.1.3 OPRM1 DNA methylation and age 145 7.3.2 Gender and methylation 146 7.3.3 Intra-individual gene methylation tissue differences in opioid associated deaths 147 7.3.4 Intra-individual gene methylation tissue differences in controls 149 7.3.5 Opioid exposed tissues v non-opioid exposed tissues 151 7.3.6 Genetic variations v blood drug concentrations 154 7.4 Discussion 157 7.4.1 Methylation of ABCB1/MDR1 gene 159 7.4.2 Methylation of OPRM1 gene 160 7.5 Conclusion 162 CHAPTER 8.0 PILOT TRIAL: PERSONALISING OPIOID THERAPY FOR CANCER PAIN RELIEF.