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3-2017 Genetic Polymorphism of -1A2 (CYP1A2) and N-Acetyltransferase-2 (NAT2) Among Emiratis Mohammed Majed Al-Ahmad

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United Arab Emirates University

College of Medicine and Health S ciences

GENETIC POLYMORPHISM OF CYTOCHROME P450-1A2 (CYP1A2) AND N-ACETYLTRANSFERASE-2 (NAT2) AMONG EMIRATIS

Mohammad Majed AI-Ahmad

This dissertation is submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy

Under the Supervision of Professor Salim Bastaki

March 2017 ii

Declaration of Original Work

I, Mohammad Majed AI-Ahmad, the undersigned, a graduate student at the

United Arab Emirates University (UAEU), and the author of this dissertation entitled

"Genetic Polymorphism of Cytochrome P450-1A2 (CYP1A2) and N­

Acetyllransferase-2 (NA T2) Polymorphism among Em ira tis" hereby, solemnly declare that this dissertation is an original research work that has been done and prepared by me under the supervision of Professor Salim Bastaki, in the College of

Medicine and Health Sciences at UAEU. This work is entirely original and has not previously been presented or published, or formed the basis for the award of any academic degree, diploma or a similar title at this or any other university. Any materials borrowed from other sources (whether published or unpublished) and relied upon or included in my dissertation have been properly cited and acknowledged in accordance with appropriate academic conventions. I further declare that there is no potential conflict of interest with respect to the research, data collection, authorship, presentation and/or publication of this dissertation.

Student's Signature: _---'-\-_======-�---:==�_ Date: l Y: Is-I2!JJr . iii

Copyright © 2017 Mohammad Majed AI-Ahmad All Rights Reserved iv

Advisory Committee

1) Advisor (Committee Chair): Professor Salim Bastaki

Title: Professor of Pharmacology and Therapeutics

Department of Pharmacology

College of Medicine and Health Sciences

2) Co-advisor: Professor Bassam Ali

Title: Professor of Molecular and Genetic Medicine

Department of Pathology

College of Medicine and Health Sciences

Member: Professor Yousef Abdulrazzaq 3) Title: Professor of Paediatrics

Department of Paediatrics

College of Medicine and Health Sciences v

Approval of the Doctorate Dissertation

This Doctorate Dissertation is approved by the following Examining Committee

Members:

1) Advisor (Committee Chair): Professor Salim Bastaki

Title: Professor of Pharmacology

Department Pharmacology

College: Medicine and Health Sciences

' Signature ___=-__ S.L.- V"""' -'='!==------Date :2'1-3 -2[/7 1

2) Member: Professor Bassam Ali

Title: Professor of Molecular and Genetic Medicine

Department: Pathology

College: Medicine and Health Sciences

Signature Date ?#3/ 2 .:.. I f ___f7.p;-I} +-M�_ ' � I ______

Member: Assistant Professor Suraiya Anjum Ansari 3)

Title: Assistant Professor of Biochemistry

Department Biochemistry

College: Medicine and Health Sciences

h ::8 -rl Signature ___ S Date c:.,..._§;::::::z:::::.....:--=---=-====-== ==------Member (External Examiner): Professor Ann 4) Katherine Daly

Title· Professor of Pharmacogenetics

Department:lnstitute of Cellular Medicine

Institution: Newcastle University, UK

Signature � Date L:=:j( '3J 1"7.- •

This Doctorate Dissertation is accepted by:

Acting-Dean of the College of Medicine and Health Sciences: Professor Ruth

Langer (

_ __/ 7 \ o( ( (1. Signature -->. \ Date 1 ':::1_-_+-'\Jr---______

Dean of the College of the Graduate Studies: Professor Nagi T. Wakim

Signature (�:� Date I 'ik /201.7

Copy 7 of ! s= vii

Abstract

Brief introduction: There are limited studies on CYP1A2 and NAT2 polymorphisms among Emiratis. Aims: This study aims to determine CYP1A2 and NAT2 alleles and genotypes and correlate these genotypes with caffeine metabolism phenotypes among Emiratis. Methods: After obtaining informed consent, five hundred and eighty one non-smoker subjects were given 300ml of caffeinated soft drink and were asked to provide a buccal swab and a urine sample two hours later. TaqMan Real

Time PCR, PCR-RFLP, DNA sequencing were performed to determine CYP1A2 and NA T2 alleles and genotypes. Phenotyping was carried out by analysing the caffeine metabolites using HPLC analysis. Results: We found that and 1.4%, 16.3% 82.3% of the recruited subjects were slow, intermediate and rapid CYP1A2 metabolisers, respectively. Only 1.4% of the subjects were homozygotes for CYP1A2 mutant alleles while 16.1 % were heterozygotes and 82.5% were homozygotes for the CYP1A2 wild type genotype. Therefore, the frequency of the wild type genotype CYP1A2*1A1*1A was 0.825 followed by CYP1A2*1A/*1C and

CYP1A2*1A1*1K, with frequencies of 0.102 and 0.058, respectively. The degree of phenotype/genotype concordance was 81.6%. The CYP1A2*1C/*1C and

CYP1A2*31*3 genotypes showed the lowest phenotype status. With regards to

NAT2, we found that 78.5%, 19.1 % and 2.4% of the subjects were slow, intermediate and rapid NAT2 acetylators, respectively, with 77.4% being NA T2 homozygote or heterozygote for mutant alleles and 18.4% and 4.2% were heterozygote and homozygote for the NA T2 wild type genotypes, respectively. The most common genotypes found were NA T2*581*78, NA T2*58/*6A, NA T2*78/*148 and NAT2*4/*58 with frequencies of 0.255, 0.135, 0.105 and 0.09, respectively. The degree of phenotype/genotype concordance was equal to 96.2%. The

NA T2*6A/*6A , NA T2*6A/*78 , NA T2*78/*78, NA T2*5A/*58 and NA T2*5A/*5A viii genotypes were found to be associated with the lowest 5-Acetylamino-6-

Formylamino-3-MethyluraciI/1 -Methylxanthine (AFMU/1 X) ratios. Significant contributions: The majority of the studied Emiratis are slow NAT2 acetylators with implications for the prescription of medications that are metabolised by this .

In addition, a small percentage of Emiratis have slow CYP1A2 enzyme activity which again should be taken into consideration when prescribing medications that are partially metabolised by this enzyme. In Emirati population, the frequency of the

CYP1A2*1A and NAT2*5 alleles were the highest relative to other alleles frequencies. Moreover, Individuals who carried NA T2*6A/*6A, NAT2*6A/*7B,

NAT2*7BI*7B, NAT2*5A/*5B, NAT2*5A/*5A, CYP1A2*1CI*1C or CYP1A2*3/*3 genotypes might be at high risk of toxicity with some drugs and some diseases compared to others as these genotypes are associated with the slowest phenotype status. Consequently, ge netic testing is recommended prior to prescribing medications that are largely metabolized by CYP1A2 or NAT2. Gap filled: This is the first detailed study of CYP1A2 and NA T2allel es and genotypes among Emiratis.

Keywords: Cytochrome P450-1A2 (CYP1A2) polymorphisms, N-acetyltransferase-

2 (NAT2) polymorphisms, Emiratis genetics, drug metabolism. ix

Title and Abstract (in Arabic)

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*41*5BJ *7B1*14BJ *5B1*6A15B1*7B ��\ w�' I ui �i �J .NAT2 J!l \ � Ji.. J *5B1*6A J *5B1*7B 0.225 JJ� 0.105 J,J)l} 0.135 J!l)U �)r\ �4 i.JL::ij1 ys'il

NAT2 *6A1*6A, NAT2 *6A1*7B, NAT2 *7BI*7B, NAT2 *5A1*5B and)

. �l.....:.. - .L 'tij 1 � 1 �. I . 0:!.Y'" uc ,�\..b.\1 b...lll' \ J . jA (NAT2 *5A/*5A � ...r . �. i <::-' l.P C'"� .

NAT2 *6A1*7B, NAT2 *7BI*7B, NAT2 *5A1*5B, NAT2 *5A/*5A, CYP1A2 *1CI*1 C

.• NAT2) CYP1A2 A..h....1.J-! ��)1\ ty:ill � J.J� "-.cy u-o JJ'iI6:...,J\ I� ufo I�J . �

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Acknowledgments

I express my deep sense of gratitude to Professor Salim Bastaki and

Professor Bassam Ali. Their dedication and keen interest above all their overwhelming attitude to help their students had been solely and mainly responsible for completing my work.

I thank profusely Professor Salim Bastaki, Professor Bassam Ali and

Professor Yousef Abdulrazzaq for their kind help, cooperation, invaluable assistance, supportand guidance throughout my study period.

l owe a deep sense of thanks to the chairman, all members of the

Department of Pharmacology, Pharmacology and Pathology lab staff at the United

Arab Emirates University for assisting me in my studies and research. My special thanks are extended to Ms Naheed Amir who helped in ordering and maintaining the required materials for my studies. Nevertheless, my thanks extended to others who provided help and supported me during my study particularly Mr Dhanasekhar, Mr.

Azim Ullah Shamsulislam and Ms Anne John.

I would like to convey thanks to the College of Medicine and Health Sciences for giving me this opportunity to get my PhD degree.

Finally, I acknowledge with thanks to my family and friends for their constant encouragement during my research period. xii

Dedication

To my family who helped and supported me from day one, to my colleagues, friends

and supervisors; Professor Salim Bastaki, Professor Bassam Ali and Professor

Yousef Abdulrazzaq, who guided and helped me undertake this project.

I thank them all for the support and help they offered me throughout my journey in

accomplishing this thesis/dissertation and getting my PhD degree. xiii

Table of Contents

...... , ...., ...... " ..... Title ...... ,-, ...... ' ...... ,

Declaration of Original Work ...... '" ... '" ...... ii

Copyright ...... iii

...... '" -,...... -, Advisory Committee ...... iv

...... , Approval of the Doctorate Dissertation ...... , ...... v

Abstract ...... vii

...... Title and Abstract (in Arabic) ...... ,- ...... IX

Acknowledgements ...... '" ...... '" ...... xi

Dedication ...... ,...... xii

T able of Contents ...... xiii

List of Tables xvii

List of Figures xix

List of Symbols, Acronyms, Abbreviations ...... xxii

List of Glossary ...... xxv

Chapter 1: Introduction 1

1. 1 Overview ...... 1

1.1.1 Background 1

1.1.2 Factors Influence Drug Metabolism ...... 2

1.1.2.1 Genetic Polymorphism ...... 2

...... ,-, ...... 4 1.1 .2.2 Diseases .., ......

...... , ...... 4 1.1.2.3 Age ..." ......

1.1.2.4 Concomitant Drugs ...... 4

1.1.3 Cytochrome P450 (CYP450) .. , ...... , ...... 6

1.1.3. 1 Cytochrome P450 Family ...... 7 xiv

1 1.4 CYP1A2 Isoform ...... , ...... 9

-,. ,-, ...... 1.1.5 Acetylation ...... 10

. , ...... ,- ...... 1.2 Statement of the Problem ...... , ...... 12

...... ,-, ...... 1.3 Literature Review ...... 15

... , . ' ...... '" - ..... '" ...... 1.3.1 CYP1A2 ...... - ...... ,-, ...... -,.. ,- ...... 15

1.3.1.1 Localization, Structure and Expression ...... 15

1.3.1.1.1 The CYP1 A2 Gene Family ...... 15

1.3.1.1.2 Reaction Mechanism, Structural Features and Functional

Relevance of CYP1 A2 ...... 16

1.3.1.1.3 CYP1A2 Regulation ...... 18

1.3.1.1.4 CYP1A2 Variants and its Molecular Epidemiology ...... 18

1.3.1.1.5 CYP1A2 Important Haplotypes ...... 24

1.3.1.1.6 CYP1 A2 Alleles NomenclatureNariant Annotations...... 27

1.3.1.1.7 CYP1A2 Genotype/Phenotype Relationship ...... 28

1.3.1.1.8 Clinical Impact, Drug Metabolism and Substrate

Specificity of CYP1A2 Polymorphism ....., ...... 30

1.3.2 N-Acetyltransferase-2 (NAT2) ...... 36

1.3.2.1 Gene Localization, Structure and Expression ...... 36

1.3.2.1.1 N-acetyltransferase Gene Family ...... 36

1.3.2.1.2 Reaction Mechanism, Structural Features, Variations and

Functional Relevance of NAT2 ...... 37

.. - ...... 1.3.2.1.3 NAT2 Regulation ...... - ..... - ...... 39

1.3.2.1.4 NAT2 Variants and its Molecular Epidemiology ...... 40

1.3.2.1.5 NAT2 Important Haplotypes ...... 45

1 .3.2.1.6 NAT2 Alleles Nomenciature Nariant Annotations .., ...... 45

1.3.2.1.7 NAT2 Genotype/Phenotype Relationship ...... 46 xv

1.3.2.1.8 Clinical Impact, Drug Metabolism and Substrate

Specificity of NA T2 Polymorphism ...... 48 1. 4 Potential Contri butions and Lim ...... itations of the Study ...... 58

Chapter 2: Aims and Objectives ...... '" ...... 59

'" ...... , 2.1 Aims ...... , ...... 59

.. ... - .. . 2.2 Specific Objectives ...... - ...... 59

...... Chapter 3: Methods ...... - ...... - ...... " ...... 60

3.1 Research Design ...... 60

3.1.1 Type of Study ...... '" ...... '" ...... 60

3.1.2 Setting ...... 60

- •• •••••• ••• 3.1.3 Including Criteria ••• ••• ,-, _,0 .,•• •••••••• •••••0 •••••••••••••• 0 ••••••••••••• 60

,0' _ ••••• ••• • •• 3.1.4 Exclusion Criteria ' •• •••••••• , •••••• •••••• ••• ••••••••, ••• ••• ' ••••• •••••• 60

3.1.5 Place of Study ...... 60

3.1.6 Study Duration I Period of Study 61

3.1.7 Ethics and Ethical Approvals ...... 61

3.1.8 Sampling ...... 61

3.2 Data Collection ...... 62

3.2.1 Sample Collection 63

3.2.2 Storage and Arrangement for Urine Samples and Buccal Swabs 63

3.2.3 Determination of Phenotypes 64

3.2.3.1 The Extraction Protocol of Caffeine Metabolites from Urine 64

3.2.3.2 Using HPLC Method 64

3.2.4 Determination of Genotypes 66

3.2.4.1 The Extraction Protocol of DNA from the Isohelix SK1 Buccal

Swabs ...... 66

3.2.4.2 TaqMan Real Time PCR (Polymerase Chain Reaction) .., ...... 68 xvi

3.2.4.3 Polymerase Chain Reaction - Restriction Fragment Length

Polymorphism (PCR - RFLP) ...... '" ...... 78

3.2.4.3.1 Polymerase Chain Reaction (PCR) 78

3.2.4.3.2 Restriction Fragment Length Polymorphism (RFLP) ...... 81

3.3 Data Analysis ...... 87

Chapter 4: Results ...... 88

4.1 Population Data ...... 88

4.2 Descriptive Analysis of the Recruited Sample ...... 89

4.3 HPLC Analysis ...... 90

4.4 Statistical Analysis 92

4.4.1 CYP1A2 ...... 92

4.4.1.1 CYP1A2 Genotype ...... 92

4.4.1.2 Caffeine Metabolites Ratio and CYP1A2 Phenotype Status ...... 97

4.4.2 NA T2 ...... 103

4.4.2.1 NA T2 Genotype ...... 103

4.4.2.2 Caffeine Metabolites Ratio and NAT2 Phenotype Status 113

Chapter 5: Discussions 121

5.1 CYP1A2 122

5.2 NAT2 124

Chapter 6: Conclusions ...... 131

6. 1 CYP1A2 ••• ••• •••••• •••••• •••••• , •• •••••• , ••••• eo. , ••••• ••••••••• ••••••••••••••••••••••••••••••••• 131

6.2 NAT2 ••• •••••• , •••••••• ••• -eo ,0' _, ••••••• , •• ••• ••• •••••• .••••• ••••••••••••••••••••••••••••••••••• 131

References ...... 132

List of Publications ...... , ...... " ." ...... 156

Appendices ...... , ...... , ...... 157 xvii

List of Tables

Table 1: The summary of the cytochrome P450 protein

families ...... '" ...... 8

Table 2: Frequencies of important SNPs in Human CYP1A2 Gene among

various populations ...... '" ... '" ...... 21

Table 3: CYP1A2 alleles, their references SNPs and catalytic activity based on the Human Cytochrome P450 Allele Nomenclature Committee ...... 27

Table 4: Frequency of important SNPs in human NA T2 gene ...... 42

Table 5: Frequency of other SNPs in human NAT2 gene ...... 43

Table 6: NAT2 alleles and its references SNPs and catalytic

activity ...... 45

Table 7: Effect of acetylator status on drug response and toxicity...... 57

Table 8: The expected distribution of our samples based on the distribution of UAE (Emiratis) between the Emirates...... 62

Table 9: Classification of CYP1A2 enzyme activity based on the ratio of (17MX+ 17MU)/1 37MX...... 66

Table 10: The acetylator status of NAT2 activity was classified based on the ratio of AFMU/1 MX...... 66

Table 11: Fluorescence signal correlations...... 69

Table 12: Assay 10 or design that has been used for each SNP...... 72

Table 13: Preparation of TBE solution...... 81

Table 14: The restriction cleavage for the amplificates of the PCR product...... 82

Table 15: The restriction cleavage site and its DNA fragment lengths from the restriction cleavage...... 84

Table 16: Evaluation sheet (..J or • = fragment length combination found) of the restriction with mutation sites and fragment length 86 for NA T2...... xviii

Table 17: The CYP1A2 genotype frequencies and distribution...... 94

Table 18: The characteristic features and frequencies of the various CYP1A2 haplotypes identified among Emiratis ...... 95

Table 19: The classification and the results of the CYP1A2 enzyme activity based on the ratio of (17MX+ 17MU)/1 37MX ...... 98

Table 20: (17MX + 17MU)/1 37MX molar ratios in subgroups assigned to CYP1A2 allele combinations...... 99

Table 21: (17MX + 17MU)/1 37MX ratios vs. genotypes. Suggested genotypic state: SIS homozygously slow, RIS heterozygotes intermediate, R/R homozygously rapid...... 99

Table 22: NAT2 diplotypes and genotypes in the study group. Comparison between genotypes reconstructed by PHASE and actual genotypes obtained by gene mapping ...... '" ...... 107

Table 23: The haplotype frequencies of NAT2...... 109

Table 24: Details of the results of the 6 samples that has been analysed using DNA sequencing...... 111

Table 25: The classification and the results of the acetylator status of NAT2 activity and its sub-classification based on (AFMU/1 MX) ratio...... 113

Table 26: AFMU/1X molar ratios in subgroups assigned to NAT2 allele combinations ...... , ...... , ...... ' ...... , ... .. 115

Table 27: AFMU/1X ratios vs. genotypes, based on the last update of NAT nomenclature scheme published by The Arylamine N­ acetyltransferase Gene Nomenclature Committee according to consensus guidelines (Hein OW et al 2008). Genotypic acetylator state: SIS homozygously slow, RIS heterozygotes, RlR homozygously rapid...... 116 xix

List of Figures

Figure 1: The process of drug metabolism through phase I, II and . 111...... 2

Figure 2: Spectrum of P450 ...... " ...... 6

Figure 3: Structure of cytochrome P450 ...... 7

Figure 4: Illustrates the process of acetylation of ...... 12

CYP1A2 gene structure ...... Figure 5: 17

SNPs of the CYP1A2 gene ...... Figure 6: Non-synonymous 19

Figure 7: Schematic representation of caffeine metabolites. CYP1A2 catalyzes the 3-demethylation of caffeine to paraxanthine and NAT2 catalyzes the acetylation of paraxanthin metabolites to AMFU ...... 29

Figure 8: Chromosomal location and structure of N-acetyltransferase (NA T2)

gene ...... 37

Figure 9: Structure of human NAT2 gene...... 38

Figure 10: The metabolic pathways responsible for the activation and detoxification of the drug ...... 53

Figure 11: Instruction of Iso helix SK1 buccal swabs use...... 63

Figure 12: Complementary TaqMan probe fluorescenes after it anneals to the template and after cleavage by AmpliTaq Gold DNA Polymerase UP...... 70

Figure 13: Overview of a genotyping experiment using Real Time PCR ...... ······ ··...... 73

Figure 14: The thermal cycling condition specified for our TaqMan genotyping assays for CYP1A2 ...... , ... , ...... 75

Figure 15: The Standard mode thermal cycling setting ...... 75

Figure 16: Variation in clustering due to the genotype of the target allele...... , ...... , ....., ...... 76

Figure 17: The allelic discrimination plate read document...... 77 xx

Figure 18: RT-PCR plate layout during sample running in the

system ...... 77

Figure 19: The thermocycler program for the NA T2- PCR...... 80

Figure 20: NA T2 gene structure with the mutation sites ...... 85

Figure 21: The age distribution of recruited subjects ...... 88

Figure 22: Representative chromatogram curves that are specific for caffeine metabolites with their retention time ...... 90

Figure 23: Standard Spectral conformation of 137MX, 17MX, 17MU, 1 MU,

1 MX and AFMU. The amount on X axis is in ug/ml ...... 91

Figure 24: The genotype frequencies of the studied sample - 0.014 of our subjects were homozygote for mutant alleles, 0.161 were heterozygote and 0.825 were homozygous for the wild type genotyping ...... 92

Figure 25: Allele frequencies- allele CYP1A2*1C frequency = 0.0636, allele CYP1A2*1k = 0.0287, allele CYP1A2*3 = 0.0027, allele CYP1A2*4 = 0 and allele CYP1A2*6 = O. The wild type allele frequency was found to be 0.905...... 93

Figure 26: An example of allelic discrimination plot that demonstrates a variation in clustering due to the genotyping of the targeted allele

(CYP1A2*1C) ...... ,...... 93

Figure 27: The frequency of CYP1A2 alleles among Emiratis in comparison with other populations...... 97

Figure 28: The distribution of subjects based on CYP1A2 activity (17MX+ 1 7MU)/1 37MX ratio ...... · .. ········ 98

Figure 29: The frequency of CYP1A2 poor metablolizers among Emiratis in comparison with other populations...... 100

Figure 30: The genotype frequencies- 0.774 of our subjects were homozygote or heterozygote for 2 mutant alleles genotyping, 0. 184 were heterozygote and 0.042 were homozygotes for the wild type genotyping ...... ······················ ··············· 104

Figure 31: Allele frequencies- allele NA T2*5 frequency = 0.364, allele NA T2 *6 = 0.1 29, allele NA T2*7 = 0.274 and allele NA T2*14 =0.099. The wild type allele frequency was found to be 0.134...... 104 xxi

Figure 32: Representative images of the gel documentation of the DNA fragments after amplification and restriction cleavage. bp = base pairs, w = wild type = reference sequence (RS) and m = mutant = sequence variation (SV) ...... 106

Figure 33: The frequency of NAT2 slow acetylation alleles among Emiratis in comparison with other populations...... 110

Figure 34: An example of DNA chromatogram of T -51 OC variant of sample 3 DNA ...... ,...... 111

Figure 35: The distribution of subjects based on NAT2 activity (AMFU/1 MX) ratio...... 114

Figure 36: Comparison of NAT2 phenotype and genotype in 555 116 subjects ...... Figure 37: The frequency of NAT2 poor metablolizers among Emiratis in comparison with other populations...... 117 xxii

List of Symbols, Acronyms, Abbreviations

137MX or 137X: Caffeine (1 ,3,7-trimethylxanthine)

17MU or 17U: 1,7 -dimethyluric acid

17MX or 17X: Paraxanthine (1 ,7-dimethylxanthine)

1MU or1U: 1-Methyluric acid

1MX or 1X: 1-Methylxanthine

AAMU: 5-acetylamino-6-amino-3-methyluracil

ADEC: Abu Dhabi Education Council

ADRs: Adverse Drug reactions

AFB1: Aflatoxin B1

AFMU: 5-Acetylamino-6-Formylamino-3-Methyluracil

AhR: Aromatic Hydrocarbon

Anti-TB drugs: Anti-Tuberculosis drugs

Arnt: AhR nuclear translocator

BP: Blood Pressure

CBT: Caffeine Breath Test

CI: Confidence Interval

CoA: Coenzyme A

CT solution: Capture buffer

CYP1A2: Cytochrome P450 -family "1" subfamily "A" gene "2"

Dill: Drug Induce Liver Injury

DNA: Deoxyribonucleic acid dNTPs: Deoxyribonucleotide triphosphates

GSTT1: Glutathione S-Transferase Theta 1

HPLC: High Performance Liquid Chromatography

INH: Isoniazid xxiii

LS solution: Lysis buffer

MAOB. Monoamine Oxidase B

NAPQI: N-acetyl-p-benzoquinone imine

NAT1: N-Acetyltransferase-1

NAT2: N-Acetyltransferase-2

NATP1: N-Acetyltransferase pseudogene

NFQ: Nonfluorescent quencher

ORF: Open Reading Frame

PABA: Para-Aminobenzoic Acid

PAHs: Polycyclic Aromatic Hydrocarbons

PAS: Para-Aminosalicylic Acid

PAS: Per-Arnt-Sim

PCR: Polymerase Chain Reaction

PCR-RFLP: Polymerase Chain Reaction - Restriction Fragment Length Polymorphism

PCT: Porphyria Cutanea Tarda

PD: Parkinson Disease

Per: Period (Drosophila period clock protein)

PGx: Pharmacogenetics

PK solution: Proteinase K

PK: Pharmacokinetics

SLE: Systemic Lupus Erythematosus

SMZ: Sulfamethazine

SNP: Single Nucleotide Polymorphism

SPSS: Statistical Package for the Social Sciences

Rheumatoid Arthritis RA:

TaqMan MGB: TaqMan Minor Groove Binder xxiv

TE solution: Tris and EDTA solution (Rehydration buffer)

Tm: Melting temperature

UAE: United Arab Emirates

UAEU: United Arab Emirates University

UP: Ultra-Pure

XO: Xanthine oxidase xxv

List of Glossary

Alleles One of a pair of that appear at a particular location on a particular and control the same characteristic, such as blood type or colour-blindness. Alleles are also called alleleomorphs.

BamHI A restriction endonuclease enzyme that has specific restrictive cleavage sites and detect mutation at (857G>A) site used to identified NA T2*7 (rs 1799931).

Odel A restriction endonuclease enzyme that has specific restrictive cleavage sites and detect mutations at (341 T>C) and (803A>G) sites used to identified NA T2*5 (rs 1801280) and NA T2 *12 (rs 1208) respectively.

Fokl A restriction endonuclease enzyme that has specific restrictive cleavage sites and detect mutation at (282C>T) site used to identified NA T2*13 (rs 1041983).

IUPAC The IUPAC "International Union of Pure and Applied Chemistry" nomenclature nomenclature system is a set of logical rules devised and used by organic chemists to circumvent problems caused by arbitrary nomenclature. Knowing these rules and given a structural formula, one should be able to write a unique name for every distinct compound.

Kpnl A restriction endonuclease enzyme that has specific restrictive cleavage sites and detect mutation at (481 C>T) site used to identified NA T2*11 (rs 1799929).

Mspl A restriction endonuclease enzyme that has specific restrictive cleavage sites and detect mutation at (191 G>A) site used to identified NA T2*14 (rs 1801279).

NAOPH Category groups of enzymes that use nicotinamide adenine dinucleotide phosphate (NAOP+ and its reduced form, NAOPH) dependent in redox reactions.

Polymorphism Natural variations in a gene, DNA sequence, or chromosome that have no adverse effects on the individual and occur with fairly high frequency in the general population. xxvi

Rn It is the fluorescence of the reporter dye divided by the fluorescence of a passive reference dye. It is used for fluorescence measurement.

SOS software The SOS software plots showed the results of the allelic discrimination run on a scatter plot of Allele 1 versus Allele 2. plots Each well of the 96-well plate is represented as an individual point on the plot.

Taq-I A restriction endonuclease enzyme that has specific restrictive cleavage sites and detect mutation at (590G>A) site used to identified NA T2*6 (rs 1799930). 1

Chapter 1: Introduction

1.1 Overview

1.1.1 Background

Drug metabolism is part of the xenobiotic metabolism processes that involve converting lipophilic drugs to more easily excretable hydrophilic products with each drug requiring a specific set of enzymes. The reactions included in these processes are clinically crucial in terms of optimal therapeutic dose, drug toxicity, cancer chemotherapy and some pathogens drug resistance (Mizuno N et al 2003).

Drug metabolism consists of three phases; phase I involves certain enzymes which include mainly the cytochrome P450 (CYP450) oxidases that act by adding reactive or polar groups to the xenobiotic. Usually the modified compound will be less active than the parent compound, although with some drugs this conversion forms the more pharmacologically active agents. In some cases the modified compound can be toxic, teratogenic or even carcinogenic (Meyer JM and Rodvold

KA 1996). These modified compounds will further undergo conjugation process by transferase enzymes such as N-acetyltransferase to form polar compounds (phase

II reaction). Finally, the conjugated xenobiotics will be pumped out of the cell by the

phase III efflux xeno-transporters (Figure 1) (Homolya Let al 2003). 2

� Xenobiotic

Xeno­ transporter

� Modifying enzyme XenoconJugate Phase I Conjugation enzyme Xeno-metabollsm

Figure 1: The processes of drug metabolism through phase I, II and III

Oxidation, reduction, hydrolysis, cyclization, decyclization, and addition of oxygen or removal of hydrogen, are carried out by mixed function oxidases

"CYP450 like CYP1A2" in phase I reaction (Akagah B et al 2008). Acetylation, glucuronidation, glutathione conjugation, glycine conjugation, methylation and sulphation are example of the pathways in phase II reaction (Liston HL et al 2001).

1.1.2 Factors Influencing Drug Metabolism

1.1 .2.1 Genetic Polymorphism

Genetic variation is an essential factor that contributes to interindividual variability in drug biotransformation. Genetics variations, termed genetic polymorphism, arise due to the presence of various combinations of alleles at the same chromosomal locus and in certain cases they are linked to inherited traits. In this context, the ability to metabolize a drug is a trait that is largely determined genetically. The definition of a polymorphism in pharmacogenetics is the availability 3 of at least genetic 2 groups within a population with distinctly different abilities to metabolize a drug (Poulsen HE and Loft S 1992, Hedrick P 201 1).

Genetic differences in drug metabolism are the result of genetically based variation in genes that code for enzymes and proteins responsible for the metabolism or transport of the drug. In polymorphisms, the genes show variation

(contain abnormal pairs or multiples or abnormal alleles) leading to altered enzyme activity. Differences in enzyme activity occur at different rates according to racial group. Genetic changes may inactivate or reduce enzyme activity leading to an increase in the level of the substrate drug. Genetic duplication may increase enzyme activity resulting in lower levels of the substrate drug concentration. (Poulsen HE and Loft S 1992).

Genetic polymorph isms of drug-metabolizing enzymes give rise to distinct subgroups in the population that differs in their ability to perform certain drug biotransformation reactions. Polymorph isms are generated by mutations in the genes for these enzymes, which cause decreased, increased, or absent enzyme expression or activity by multiple molecular mechanisms. Individuals can be classified into three main phenotypic groups: extensive, intermediate and poor

metabolizers. Poor metabolizers often have higher risk of adverse effects including toxicity (Meyer JM and Rodvold KA 1996).

Generally, poor metabolizers have two defective alleles (e.g.: CYP206*41*5

and CYP206*41*4) or combination of alleles including one resulting in no enzyme

(e.g.: CYP206*5 and CYP206*4 deletion).

Intermediate metabolizers on the other hand, are heterozygous "having only

one wild type allele and one defective allele". Normal metabolizers carry wild type 4

alleles (e.g.: CYP206*11*1). Wild type alleles encode genes for normal enzyme

function.

The combination of alleles encoding the gene determines the activity and

effectiveness of the enzyme. The overall function of the enzyme is the phenotype of

enzyme function. Phenotype is defined as the observable physical or biochemical

characteristics determined by both genetic makeup and environmental influences

(Meyer JM and Rodvold KA 1996).

1.1.2.2 Diseases

Infection, hepatitis, alcoholic liver disease, biliary cirrhosis, hepatocarcinoma

and other forms of impaired liver functions can lead to decreased drug

biotransformation and the degree of impairment is a function of the severity of the disease (Meyer J.M. and Rodvold K.A. 1996).

1.1 .2.3 Age

The ability to metabolize a drug is also influenced by the age of the

individual. For examples, very young infants do not have a mature enzyme system.

In addition, decreases in liver mass, hepatic enzyme activity and hepatic blood flow

are more concomitant with the elderly (Meyer JM and Rodvold KA 1996).

1.1 .2.4 Concomitant Drugs

The most common causes that affect drug biotransformation reactions are

induction and inhibition of cytochrome P450 enzymes and thus agents that promote

these processes have impact on drug metabolism as briefed in the following

sections (Meyer JM and Rodvold KA 1996). 5

A- Induction

Induction is an increase in enzyme activity that is associated with exposure to drugs. This can occur when a drug alters the biotransformation of co-administered drugs either by the same enzyme pathway or through an alternative pathway.

However, inducers are usually specific for a given cytochrome P450 family.

Sometimes a drug can induce its own biotransformation in addition to that of other agents (Meyer JM and Rodvold KA 1996).

The main factors that affect the time course of enzyme induction onset and offset are plasma concentration of the inducer and the half-life of enzyme production and degradation. The effect of induction can be seen within the first few days of therapy (Oossing M. et al 1983).

B- Inhibition

One of the most common mechanisms of inhibition is competitive inhibition and occurs when two or more drugs compete for the same enzyme, which becomes clinical significant based on the relative concentrations of the drugs, as well as, a variety of other patient specific factors (Meyer JM and Rodvold KA 1996).

The binding can be either irreversible or reversible with the heme-binding site of the enzyme and inhibits other drugs from binding, which leads to loss of function that activity can only be restored by synthesis of new enzymes, which may take several days (Meyer JM and Rodvold KA 1996).

More complex mechanisms of inhibition can occur where some drugs

undergo metabolic activation by the cytochrome P450 system to inhibitory products

that could result in relatively stable complexes with cytochrome P450. In this

scenario the cytochrome is held in an inactive state that can be clinically significant. 6

Additionally, the interaction involves narrow therapeutic drugs which could increase the risk of toxicity (Meyer JM and Rodvold KA19 96).

Unlike induction enzyme, inhibition usually begins with the first dose of the inhibitor. The inhibition reaches the maximum level when the inhibitor reaches steady state (four to five half-lives). Similarly, the time needed for the interaction to resolve depends on the half-lives of the drugs involved (Dossing M et al 1983).

1.1.3 Cytochrome P450 (CYP450)

Cytochrome P450 enzymes are composed of a large superfamily of heme- thiolate proteins responsible for the metabolism of a wide variety of both exogenous and endogenous compounds (Degtyarenko KN 1995). In 1955, it was discovered in rat liver microsomes and they were characterized by an intense absorption band at 450 nm in the presence of carbon monoxide (Vercruysse A

1997) (Figure 2).

o.3 r-----.-----�----.-----_r----_.----�

------�---� � . 2L- � L- �-- � 0 0 410 430 450 470 49 5 1 Wavelength (nm) Figure 2: Spectrum of P450

The cytochrome P450 (CYP) mixed function monooxygenases are located

on the smooth endoplasmic reticulum of cells throughout the body and are present 7 at highest concentrations in the liver (hepatocytes) and small intestine. These enzymes are largely re sponsible for phase I xenobiotic metabolism (Figure 3) (Buck ML 1997).

Cytochrome P450sp

POB code: 3AWM

Figure 3: Structure of cytochrome P450

1.1.3.1 Cytochrome P450 Family

Almost 12 cytochrome P450 gene fa milies have been reported in the . However, only 3 families, the cytochrome P450 1, 2 and 3 (CYP1, CYP2 and CYP3) are involved in the majority of drugs biotransformations (Meyer JM and

Rodvold KA 1996). Based on amino acid sequence similarities the enzymes are divided into families, and each family can be further segregated into subfamilies, which are identified by capital letters following the family designation (for example

CYP1 subfamily A is designated CYP1A). The most important and clinically relevant cytochromes are CYP3A4, CYP206, CYP1A2, CYP2C9, CYP2C19 and CYP2E1

(Martin J and Fay M 2001). In total, humans ha ve 57 genes and more than

59 pseudogenes divided into 18 families of cytochrome P450 genes and 43 subfamilies as shown in table 1 (Nelson DR et al 2004). 8

Table 1- The summary of the cytochrome P450 protein families

Family Members Gene Names

subfamilies, genes, CYP1 3 3 CYP1A1, CYP1A2, CYP1B1 1 pseudogene

CYP2 13 subfamilies, 16 genes, CYP2A6, CYP2A 7, CYP2A 13, CYP2B6, CYP2C8, C 16 pseudogenes YP2C9, CYP2C18, CYP2C19, CYP206, CYP2E1, CY P2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2 W1

1 subfamily, genes, 2 CYP3 4 CYP3A4, CYP3A5, CYP3A 7, CYP3A43 pseudogenes

6 subfamilies, 12 genes, CYP4 CYP4A 11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, C 10 pseudogenes YP4F8, CYP4F1 1, CYP4F12, CYP4F22, CYP4 V2, CYP4X1, CYP4Z1

CYP5 1 subfamily, 1 gene CYP5A 1

CYP7 2 subfamilies, 2 genes CYP7A 1, CYP7B1

CYP8 2 subfamilies, 2 genes CYP8A 1, CYP8B1

CYP1 1 2 subfa milies, 3 genes CYP1 1A1, CYP1 1B1, CYP11B2

CYP17 1 subfamily, 1 gene CYP1 7A1

CYP19 1 subfamily, 1 gene CYP19A 1

CYP20 1 subfamily, 1 gene CYP20A 1

CYP2 1 2 subfamilies, 1 gene, 1 CYP2 1A2 pseudogene

CYP24 1 subfamily, 1 gene CYP24A 1

CYP26 3 subfamilies, 3 genes CYP26A 1, CYP26B1, CYP26C1

CYP27 3 subfa milies, 3 genes CYP27A 1, CYP27B 1, CYP27C1

CYP39 1 subfamily, 1 gene CYP39A 1

CYP46 1 subfamily, 1 gene CYP46A 1

CYP51 1 subfamily, 1 gene, 3 CYP5 1A1 pseudogenes 9 1.1.4 CYP1 A2 Isoform

Each of the isoforms has wide substrate specificity, but each has its own specific substrate characteristics. These isoforms have differing regulatory mechanisms to control their activity. The regulatory mechanisms involve chemicals which Induce or inhibit the enzyme. For example, GYP1A2 metabolises some carcinogenic tars in cigarette smoke and is induced by these chemicals. Members of other GYP gene families are induced by drugs such as barbiturates, anticonvulsants and rifampicin (Martin J and Fay M 2001).

As well as showing some degree of substrate selectivity, the individual isoforms also show selectivity for inhibitors. For example, sulfaphenazole is a specific inhibitor of GYP2G9 whereas quinidine is a potent and selective inhibitor of the isoform GYP2D6 (Martin J and Fay M 2001).

All isoforms will show some polymorphism. The frequency of these polymorphisms differs markedly between ethnic groups. These genetic differences mean some people have an enzyme with reduced or no activity. Patients who are

'slow metabolisers' may have an increased risk of adverse reactions to a drug metabolised by the affected enzyme (Martin J and Fay M 2001).

Variety of cytochrome P450 enzymes could be found in a single hepatocyte.

An individual enzyme of cytochrome P450 may be able of metabolizing many different drugs; however, a given drug may be primarily metabolized by a single enzyme (Meyer JM and Rodvold KA 1996).

Drug interactions involving the cytochrome P450 system may not necessarily

be clinically significant. There are several factors that help predict whether or not a

drug interaction will be clinically significant. These include pharmacokinetic,

pharmacodynamics, the wide variability of patient response to the same drug, 10 concomitant medical illness and factors relating to the route and timing of administration may also be important (Dresser GK et al 2000).

Cytochrome P450 1A2 (CYP1A2), is one of the cytochrome P450 mixed­ function oxidase system and is responsible for the metabolism of xenobiotics in the body and involved in the synthesis of cholesterol, steroids and other lipids (Nelson

DR et al 2004).

In humans, the CYP1A2 enzyme is encoded by the CYP1A2 gene. This cytochrome P450 enzyme is localized to the endoplasmic reticulum and its expression is induced by some polycyclic aromatic hydrocarbons (PAHs), some of which are found in cigarette smoke. It is of clinical importance due to the huge number of drug interactions associated with its induction and inhibition. Crucially, it is able to metabolize some PAHs to carcinogenic intermediates. Other xenobiotic substrates for this enzyme include caffeine, aflatoxin 81, and acetaminophen

(Nelson DR et al 2004).

Caffeine is considered to be a model substrate for this enzyme due to its common consumption and safety. CYP1A2 enzyme activity may be altered by environmental factors including cigarette smoking , charbroiled meat, vegetables such as cabbages, cauliflower, broccoli and cruciferous and a number of drugs including rifampicin, phenobarbital and omeprazole (Runge 0 et al 2000).

1.1.5 Acetylation

In the IUPAC (International Union of Pure and Applied Chemistry) nomenclature, acetylation or ethanoylation is "a chemical reaction that describes the

adding of an acetyl functional group into a chemical compound". The process of

acetylation is essential in protein formation, drug biotransformation, regulation of

deoxyribonucleic acid (DNA) and other genetiC elements using histone acetylation. 11

In addition, acetylation mediated by acetyltransferase enzymes has been shown to be important in cancer and other human diseases (Robert K et al 2003 and Zhao SF et al. 2008).

N-terminal Acetylation

N-terminal acetylation is one of the most common co-translational covalent modifications of proteins in eukaryotes and it is crucial for the regulation and function of different proteins (Choudhary C et al 2009, Fritz KS et al 2012). N­ terminal acetylation plays an important role in the synthesis, stability and localization of proteins (Robert K et al 2003 and Zhao SF et al 2008). About 85% of all human proteins and 68% in yeast are acetylated at their No-terminus (Van DP et al 201 1).

Acetylation is an essential route of xenobiotics metabolism involving an aromatic amine (R-NH2) or a hydrazine group (R-NH-NH2), which are converted to aromatic amides (R-NH-COCH3) and hydrazides (R-NH-NH-COCH3), respectively as well as for a number of known carci nogens present in the diet, ciga rette smoke and the environment (Starheim K et al 201 2).

N-terminal Acetylation is catalyzed by a set of enzyme complexes, the N­ terminal acetyltransferases (NATs). The reaction pathway is catalyzed by two cytoplasmic acetyltransferases, N-acetyltransferase Type I (NAT1 ) and N­ acetyltransferase Type \I (NAT2). These enzymes are located in the liver, lung, spleen, gastric mucosa, red blood cells (RBCs) and lymphocytes and the presence of co-factor acetyl coenzyme A is essential (Liston HL et al 2001). NATs transfer an acetyl group from acetyl-coenzyme A to the a-amino group of the first amino acid

residue of the protein as shown in figure 4 (Starheim K et al 2012). 12

Nfl -terminal acetytransferases Polypeptide 0 II e (NATs) �C � .. NH3

I R N-terminal Ac-CoA CoA amino group N·terminal acelytated Polypeptide

Figure 4: Illustrates the process of acetylation of proteins

The genes encoding the two NAT proteins were first identified by Grant et al

(1991) who showed that each component consists of an intronless Open Reading

Frame (ORF) of 870 base pairs. The two genes are 87% homologous and are located at 8p22, a chromosomal region commonly deleted in some human cancers

(Grant OM et al 1991, Starheim K et al 2012).

The acetylation polymorphisms in NA T2 were observed in tuberculosis patients who received isoniazid and reported toxic side effects. This has been discovered over 60 years ago (Hughes HB 1954). In fact, the polymorphism was named the "isoniazid acetylation polymorphism" for several years until the significance of polymorphisms in the metabolism and disposition of other drugs and chemical carcinogens was fully appreciated (Weber VVW and Hein OW 1985).

1.2 Statement of the Problem

Cytochrome P450s (CYPs) are responsible for the metabolism of a wide variety of clinically, physiologically and toxicologically important compounds

(Guengerich FP. 2001). CYP families 1, 2 and 3 are responsible for 70-80% of

phase I metabolism of clinically used drugs (Eichelbaum M, et al 2006) and are

included in the biotransformation of a huge number of xenobiotics. Biotransformation

can turn in bioactivation of pre-carcinogens to carcinogenic and drugs to toxic 13 metabolites or other toxic effects. The majority of CYP-mediated xenobiotic metabolism is carried out by polymorphic enzymes (Guengerich FP 2006).

Nevertheless, many reports showed that both genetic mutations and environmental factors influence the elimination of drugs that are metabolized by

CYP1A2 (Ingelman-Sundberg M et al 2007, Zhou SF et al 2008, Gunes A and Dahl

ML 2008 and Zhou SF et al 2009a).

Plasma levels of a drug can vary between individuals (Ingelman-Sundberg M

2001 b and Cascorbi I 2006). In fact, genetic factors are one of the main factors that play important role in the differences between individuals in the response and susceptibility to drugs and xenobiotics (Ingelman-Sundberg M 2001 a). Variability among individuals in drug metabolism can result in either reduce drug efficacies or increase risk of side effects, which are phenomena that may influence drug therapy and development.

Several adverse drug reactions reports related to specific drugs metabolic pathways that are catalysed by polymorphic CYP enzymes have been reported. For that, the complete understanding of the nature of CYP polymorph isms is crucial to gain a comprehensive view of the inter-individual differences in chemical exposure, adverse drug effects and toxicity (Ingelman-Sundberg M 2002).

CYP1A2 is an important enzyme that bioactivate a number of

procarcinogens and some natural compounds such as aristolochic acids. In addition,

this enzyme also metabolizes a number of essential endogenous compounds (Wang

8 and Zhou SF 2009). Several studies implicated CYP1A2 polymorph isms in cancer

susceptibility (Suzuki H, et al 2008 and Altayli E, et al 2009) and other diseases

(Pruitt KD et al 2012). 14

On the other hand, some studies have recorded that there is a strong association between the acetylation status and the risk of many diseases (Evan DA

1984, Clark DW 1985, Evan DAP 1993).

NAT enzymes polymorph isms have been suggested to be associated with risk of cancer, treatment responses and treatment resistance due to their roles in the activation or deactivation of xenobiotics. NAT2 enzymes have been implicated in chemical carcinogenesis pathways. For instance, several studies had examined the widespread occurrence of NA T2 genes polymorph isms in the population that have contributed to individual risk of having disease (Weber WW and Hein D W. 1985,

Sim E et al 2008, Stanley LA and Sim E 2008, Sim E et al 2012, McDonagh EM et al

2014).

As mentioned earlier, NAT2 is a polymorphic enzyme that has essential roles in the deactivation or activation of numerous xenobiotics in humans. Based on the expression of its isoenzymes in the liver, the genetic variants of NA T2 have been correlated with drug metabolism, response and toxicity. In addition, drug metabolic rates and susceptibility to drug toxicity have varied according to acetylation phenotypes status which is conferred by NA T2 genotypes. More studies are crucial to help measure the clinical significant of NA T2 to determine the patient's dosage for maximum treatment efficacy and to avoid drug toxicity (Weber WW and Hein

DW 1985, Sim E et al 2008, Stanley LA and Sim E 2008, Sim E et al 2012,

McDonagh EM et al 2014).

UAE Population

Woolhouse et al (1997) who had conducted one study among Emiratis from the eastern region of Abu Dhabi Emirate determined the Polymorphic N­

acetyltransferase (NA T2) genotypes in 106 unrelated Emiratis by PCR-RFLP

analysis. This is the first study conducted to overview CYP1A2 and NA T2 15

polymorphism among nationwide Emiratis sample and to target the determination of

the alleles and genotype frequencies and their correlation with the phenotyping in

this population.

1.3 Literature Review

1.3.1 CYP1A2

1.3.1.1 Gene Localization, Protein Structure and Expression

A complete understanding of the gene location, structure, variation and expression of CYP1A2 is crucial to gain a comprehensive view of the inter-individual differences in chemical exposure, adverse drug effects and toxicity (Ingelman­

Sundberg M 2002).

1.3.1.1.1 The CYP1A2 Gene Family

CYP1A2 (Cytochrome P450, Family 1, Subfamily A, Polypeptide 2) is a protein coding gene. This gene encodes one of the cytochrome P450 superfamily of enzymes. The transcript from this gene includes four Alu sequences flanked by direct repeats in the 3' untranslated region. In liver microsomes, CYP1A2 enzyme

plays a role in an NADPH-dependent electron transport pathway. It oxidizes a

number of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics. It is most active in catalyzing 2-hydroxylation. Caffeine is metabolized

primarily by cytochrome CYP1 A2 in the liver through an initial N3-demethylation.

Moreover, it bioactivates the carcinogenic aromatic and heterocyclic amines. It

catalyzes the N-hydroxylation of heterocyclic amines and the O-deethylation of

phenacetin (Pruitt KD et al 2012).

The CYP1A2 gene is presented in a cluster with CYP1A 1 and CYP1B1 on

chromosome 15 particularly, in the long arm reg ion q24.1. (Zhou SF et al. 2009a) 16 CYP1A2 is one of the major CYPs in human liver (-13-1 5%) compared to 2% with

CYP206 (Shimada T et al 1994).

Moreover, 60-fold interindividual variation has been reported in CYP1A2 activity (Shimada T et al 1994 and Nakagawa K et al 2002). Also, more than 15 and

40-fold variation in mRNA and protein expression levels have been detected in the human liver (Ikeya K et al 1989 and Guengerich FP et al 1999).

The genetic component of variation in CYP1A2 activity is almost 75% and the remaining is related to environmental factors, such as smoking (induction), oral contraceptive use in women (inhibition) , and others (Rasmussen BB et al 2002).

However, in 2010 Klein et al provided a pathway-based analysis in human liver samples which demonstrated that the genetic variation of CYP1 A2 activity accounted for 42, 38, and 33% of the catalytic activity, protein expression, and mRNA levels, respectively (Klein K et al 2010). In view of the predominant role of

CYP1A2 in activation of toxic xenobiotics compared with its metabolism of prescription drugs, there are a lot of epidemiological reports testing the role of variant CYP1A2, metabolism of procarcinogens, and cancer risk (Zhou SF et al

2009a).

1.3.1.1.2 Reaction Mechanism, Structural Features and Functional Relevance of CYP1A2

CYP1A2 and CYP1A 1 share a 5'-flanking region of approximately 23 kb, which includes shared regulatory elements, although the genes are positioned back to back and transcription occurs in opposite directions (Ueda R et al 2006). The

CYP1A2 gene spans around 7.8 kb and comprising seven exons and six introns, the first exon 55-bp-long noncoding exon. CYP1A2 is a 515-residue protein with a

molecular mass of 58 294 Oa (Zhou SF et al 2009a). 17

The CYP1A1 and CYP1A2 genes are split by a 23-kb segment that includes no open-reading frames (Corchero J et al 2001). Between CYP1A2 and CYP1A1, exons 2, 4, 6, and particularly 5 are strikingly maintained in both nucleotides and total number of bases (Figure 5) (Ikeya K, et al 1989).

2 Exon Exon Exon Exon Exon Exon Exon 1 (first expressed) 3 4 5 6 7

Figure 5: CYP1A2 gene structure (Sachse et al 1999)

Human CYP polymorph isms include mutations in both non-coding regions

(promoters. introns) and protein-coding sequences (exons). Mutations in non-coding regions may result in changes in the expression levels. whereas mutations in protein-coding sequences may result in alteration in the structure and catalytic propertiesof the enzyme (Zhou H. et al 2004).

There are over 100 substrates reported for CYP1A2 . including many clinically significant drugs; such as clozapine. tacrine, procarcinogens (e.g. benzopyrene and aflatoxin b1). and endogenous substrates (e.g. steroids and ) (Zhou SF. et al 2009a). However, compared with other CYPs. there have been relatively few reports of pharmacogenetics (PGx) relationships.

This can be explained by the small number of prescription drugs for which CYP1A2

is a metabolizing enzyme (9% compared with 37% for CYP3A4/5, 17% for CYP2C9.

and 15% for CYP206). Indeed for several drugs, CYP1A2 is not the sole

metabolizing enzyme. nor is it active at the rate-limiting step (Zanger UM. et al

2008). In addition. there are under-reported variants that impact CYP1A2 activity. 18

1.3.1.1.3 CYP1 A2 Regulation

CYP1A2 mar is kedly expressed in liver and is inducible in the liver, lung, pancreas, gastrointestinal tract, and brain (Jorge-Nebert LF, et al 201 0). Drug interactions have been reported many times in correlation with polymorphism of

CYP1A2 that result in alteration of drug response (Zhou SF, et al 2009a). Smoking, dietary cruciferous vegetables, polyamine hydrocarbons from grilled meat, and omeprazole and other proton pump inhibitors have been shown to increase CYP1A2 activity (Gunes A and Dahl ML 2008). Oral contraceptives, fluvoxamine, and fluoroquinolone antibiotics inhibit CYP1 A2 expression (Gunes A and Dahl ML.

2008).

Similar to CYP1A 1 and 181, CYP1A2 is primarily regulated by the aromatic hydrocarbon receptor (AhR), a l igand-activated transcription factor and a basic helix­ loop-helix protein belonging to the Per-Arnt-Sim family of transcription factors (Wang

B and Zhou SF et al 2009).

The expression regulation of CYP1A 111A2 is complex because gene transcription not only includes the AhR but also a number of transcription factors, and is potentially influenced by the actions of transcriptional coactivators and corepressors. AhR-mediated signaling pathways show a first line of defense against potentially toxic environmental contaminants. While, metabolic induction procedures by the AhR may produce highly carci nogenic metabolites, generating a link between

AhR activation and chemical carcinogenesis (Wang B and Zhou SF 2009).

1.3.1.1 .4 CYP1A2 Variants and its Molecular Epidemiology

CYP1A2 variants have been reported several times, with some implications and alteration in drug metabolism (Browning SL et al 2010). 19 More than, 177 single-n ucleotide polymorph isms (SNPs) of human CYP1A2 upstream sequence have been determined (from which 41 variant alleles (*1 B to *21) were recorded), illustrat ing the alterations on DNA sequence levels and in

some cases their epidemiological frequencies (Browning SL, et al 2010). Among the

SNPs located in seven exons, there are 22 non-synonymous that alter amino acid

sequence in exons 1-7 of CYP1 A2 (Figure 6) (Zhou SF et al 2009a).

217G>,t.

143.4A>T

136!IC >f

1217G>" I ]

'2ife T J

• UTFI 373T>"

Figure 6: Nonsynonymous SNPs of the CYP1A2 gene

The variability in liver expression of the CYP1 A2 gene reach almost 40% and

60% variability in caffeine metabolism; the most often probe drug used for CYP1A2

(Gunes A and Dahl ML. 2008). Only few variants could clearly explain the

phenotypic variability in CYP1A2 gene expression contrarily it is much more with

other drug-metabolizing CYPs (Ghotbi R et al 2007). Ghotbi et al in 2007 observed a

very low frequency of the coding sequence variants in Caucasian and Asian

populations (Ghotbi R et al 2007). Browning et al in 2010 examined CYP1A2

variation in Ethiopians, suggested that because of the overall greater incidence of

variation, including some novel presumable deleterious variants, there could be

some individuals freed from any CYP1A2 activity in this population (Browning SL, et

al 2010). 20 Nevertheless, there are several SNPs reported in the 5'-flanking region and 1 CY A2 intron of P1 (Aitchison KJ et al 2000). Aitchison et al (2010) recorded several 5'-f SNPs in the lanking region of CYP1A2, including T-3594G and G-3598T. The T-

3594G SNP has a 1.72% frequency in Caucasians (Aitchison KJ et al 2000). In the

5'-flanking region, identified SNPs include G-3860A, G-3598T, T-3594G, G-31 13A,

A-3053G, T-2847C, A-2808C, T-2667G, and -2467de1T in Japanese (Chida M et al

1999, Soyama A, et aI 2005). In intron 1, SNPs including T-739G, T-729G, G-733C,

A-587G, C-367T, C-163A, and C-99T were recorded. C-163A in intron 1 is present

re with markable frequencies in most populations tested. G-3860A, A-31 13G, _

2464de1T, T-739G, and C-729T were observed with different frequencies in various ethnic groups (Soyama A et al 2005).

Based on epidemiological studies, it has been clearly observed that there is significant ethnic variability in the distribution of common and rare CYP1A2 SNPs and haplotypes (Zhou SF et al 2009a). The G-3860A (*1C) SNP has less frequency in Caucasians than in Asians (0.21-0.25) (Chida M et al 1999). The frequency of

*1C is significantly lower in the Turkish population (0.04) (Bilgen T et al 2008) than in Japanese (0.21) (Chida M et al 1999) and Chinese (0.25), whereas it is almost equal in the Turkish and Egyptian populations (0.07) (Hamdy SI et al 2003).

The frequency of the -2467de1T (CYP1A2*10) allele is lower in Caucasians compared with Asians and Africans. The allelic frequency of the *10 allele is 4.1-

7.9% in Caucasians (Sachse C et al 2003, Skarke C et al 2005). However, the -

2467T deletion appeared at higher level frequencies of 42.0-43.8% in Japanese

(Soyama A et al 2005, Chida M et al 1999) and of 40% in Egyptians (Hamdy SI et al

2003). In Turkish, the frequency of CYP1A2*1 0 is very high (92%) (Bilgen T et al

2008). The -739T>G SNP (allele CYP1A2*1E and CYP1A2*1G) is frequent in

Ethiopians (0.10) (Aklillu E et al 2003), Saudi Arabians (0.096) (Aklillu E et al 2003), 21 and Japanese (0.082) (Chida M et al 1999). However, British (Sachse C et al 2003), German (Skarke C et al 2005), Spaniar d (Aklillu E et al 2003), Turkish (Bilgen T et al 2008), and Egyptian (Hamdy SI et al 2003) populations have a low frequency for this allele (0.0044, 0.016, 0.017, 0.01 , and 0.03, respectively) (Table 2).

Fr Table 2: equencies of important SNPs in Human CYP1A2 Gene among various populations (Nakaj ima M et al 1999, Chida M et al 1999, Hamdy SI et al 2003, Tiwari AK et al 2005, Bilgen T et al 2008, Zhou SF et al 2009b)

Ethnic group No. of G-383OA A..:I113G -2467de1T T·739G (01E. °1G. C-729T C·163A (01F. T-5347C (01B. °1H. °1G. subjects (O'e) (01D) "1J. and *11<) ( °11<) .'J. and "11<) "3, '8, *15, and ·'6) Caucasian

114 0.009 British 0.048 0004 0.333 0382

German 236 0.32

Italian 9S 004 0.24 0.33

Spanish 117 0.02 0.005

Swedish 194 0008 0.023 0.193 0.023 0.003 0.286

Swedish 1,170 0.29

Turkish 110 0.04 0.92 0.01 0.27

ASian

Chinese 27 0.22 0.09 0.50 009 0.30 0.20

Chinese 168 0.33

Japanese 159 0.21 0.42 0.08 0.61

Japanese 250 0.24 003 0.44 0.03 0.32 0.19

Korean 150 0.267 0.027 0.707 0.027 0 0.373

Saudi Arabian 173 0. 10 004

African

Egyptian 212 007 0.40 0.03 0.68

Ethiopian 173 0.10 0.03 0.40

Tanzanian 71 0.51

Tunisian 98 0.07 0.075 0. 13 0.44

Zimbabwean 143 0.43

The existence of allelic imbalance in CYP1A2 expression and the importance of epigenetic genetic variation in impacting CYP1A2 mRNA expression and enzyme activity in vitro using human liver were also reported (Ghotbi R et al 2007 ).

Epigenetic and environmental factors play a role in identifying interindividual and interethnic variability in CYP1A2 expression and enzyme activity, apartfrom genetic variation. 22 Compared to other CYP enzymes, the understanding of the

pharmacogenomic effects of CYP1 A2 variation is still at an early stage. There is

also the need to fully evaluate all other variants before assigning haplotypes

(Soyama A et al 2005, Ghotbi Ret al 2007, Browning SL et al 2010).

ImportantVar iants in CYP1A2

The most frequently studied polymorphisms are G-3860A (allele *1 C), _

2467deiT (*10), T-739G (*1E), and C-163A located in intron 1 ( *1F), which have

been initially reported in a Japanese population (Zhou SF et al 2009a). These

polymorphic CYP1A2 alleles present differences in the promoter region and are

associated with changed enzyme activity particularly, allele * 1 C, * 10, * 1 F, and * 1 K

(Zhou SF et al 2009a).

rs762551, CYP1A2: C-163A

This SNP is the most well-studied genetic variant in CYP1A2. It is the sole

variant of the CYP1A2*1 F haplotype and found with other variants in several

haplotypes (CYP1A2*1J, CYP1A2*1 K, CYP1A2*21, and others that have not been

confirmed). It is located in the intron between the noncoding exon 1 and exon 2, where the coding sequence begins and causes increase in enzyme activity (Gunes

A and Dahl ML 2008). The frequency of rs762551 :C>A varies widely with

populations: the A allele (slow) frequencies range from 0.3 to 0.39 in Asians, from

0.4 to 0.51 in Blacks or African Americans, and from 0.29 to 0.33 in Whites (Gunes

A and Dahl ML. 2008).

rs2069514, CYP1A2: G-3860A

The *1C haplotype contains G-3860A in the 5'-flanking region of CYP1A2,

originally called G-2964A (Obase Y et al 2003). 23 It was initially reported in Japanese with a frequency of 23.3% (Nakajima M

et al 1999). This allele showed decrease in enzyme activity based on the

measurement of the rate of caffeine 3-demethylation in Japanese smokers, that

could be due to the decreased inducibility and expression of the enzyme (Nakajima

M et al 1999).

The CYP1A2*1C haplotype was found at frequencies of 7% in Blacks or

African Americans, at 6-25% in Asians, and at 0.4-1 % in Whites (Nakajima M et al

1999, Hamdy SI et al 2003, Sachse C et al 2003, Tiwari AK et al 2005, Takata K et

al 2006, Ojordjevic N et al 2010). This variant was associated with a reduced rate of

caffeine demethylation in Japanese smokers but not in non-smokers. The variant

has changed a transcription factor binding site in the gene promoter; however the factor was not defined (Obase Y et al 2003).

rs1 2720461, CYP1A2: C-729T

CYP1A2: C-729T is also called CYP1A2: C-730T. This variant was shown in

vitro to affect binding of an E-twenty six transcription factor (Aklillu E et al 2003). The

*1 K haplotype was associated with 40% lower inducibility in vitro, and non-smokers

heterozygous for *1K had significantly lower CYP1A2 activity compared with the wild

type (Aklillu E et al 2003). Alkillu et al (2003) stated that this variant had lower

inducibility with 2,3,7,8-tetrachlorodibenzo-p-dioxin in human hepatoma cells and

may affect bioactivation and sensitivity to carcinogens. This could illustrate the lower

caffeine metabolism observedfor the *1 K haplotype, which is the only haplotype that

contains this variant.

CYP1A2*1 K was reported to be rare in Swedes (0.3%) and absent in

Koreans (Ghotbi R et al 2007). However, the effect of allele CYP1A2*1K could not

be found in these populations. 24 Other important variants includes rs56276455, 5347T>C (exon 4, causing

Asp348Asn and the synonymous 5347T>G (Asn516Asn)), rs72547516, 2499A>T

(exon 5, leading to Ile386Phe) and rs28399424, 5090G>T (exon 7, leading to

Arg431 Trp) which represent allele CYP1A2*3, CYP1A2*4 and CYP1A2*6, respectively. All of them are associated with decreased gene expression and enzyme activity that has been identified in the French population with frequency of

0.5-1% (Chevalier 0 et a1 2001, Allorge 0 et a1 2003, Zhou H et aI 2004).

Other CYP1A2 Variants

CYP1A2*1J (C-163A; T-739G) and *1K (C-1 63A; T-739G; C-729T, all located in intron 1) have been initially identified in Ethiopian non-smokers (Aklillu E et al 2003). The T-739G had a frequency of 3.2% in Japanese; however, the C-729T

SNP was not reported in the Japanese population (Soyama A et al 2005).

CYP1A2*2 (63C>G), mutation that causes a Phe21 Leu substitution, which was initially identified in one subject out of 157 Chinese subjects with an allele frequency of 0.32% (Huang JD et al 1999). Its functional impact is unknown. The

CYP1A2*2 allele was not found in British (n = 114) (Sachse C et al 2003) and Italian

populations (n = 500) (Pucci L et al 2007). The *5 haplotype entails 3496G>A in exon 6 resulting in Cys406Tyr (Zhou SF et al 2009a).

CYP1A2*7 (3533G>A) mutation at the splice donor site of intron 6 which has

been reported in a 70-year-old patient who had very high plasma concentrations of

clozapine when administered at normal dose. This SNP caused RNA splicing defect

and lead to a loss of CYP1A2 activity (Zhou SF et al 2009a).

1.3.1.1.5 CYP1A21mportant HapJotypes

the Human Almost 41 CYP1 A2 haplotypes have been published on

page. Twenty four Cytochrome P450 Allele Nomenclature Committee home 25 haplotypes are related to nucieotides changes in the non-coding region and 17 haplotypes are related to nucieotides changes in the coding region of the CYP1A2 protein.

There are several studies that characterize gene polymorphisms in the non­ coding region, however, only few reports describe non-synonymous protein polymorphisms. Murayama N et al (2004), Saito Y et al (2005) and Zhou SF et al

(2004) characterized most of the non-synonymous polymorphisms with a limited number of substrates.

A haplotype can refer to a combination of alleles or a set of single nucleotide polymorphisms (SNPs). This set of alleles is located on one chromosome. Thus, the alleles making up a haplotype can be located in different places on the same chromosome but they are inherited together.

An allele is one of two or more forms of the DNA sequence of a particular gene. Sometimes, different DNA sequences (alleles) may result in different gene expression or have the same result in the expression of the gene.

Many studies have focused on certain alleles as they alter the gene expression. This alteration results in changing the catalytic activity of the enzyme encoded by this gene which in turn reflects inter-individual differences in the phenotype status. The most relevant alleles are briefly described in the following section and listed in table 3.

CYP1A2*1A

This is the wild-type allele to which all variants are compared to and it is

associated with normal gene expression. 26

CYP1A2*1F

The *1 F haplotype has been reported in many studies with a changed phenotype (Ghotbi R et al 2007). This haplotype associated with increased gene expression which in turn results in increased enzyme activity. However, the phenotype effect is seen only in the availability of an inducer, such as smoking or heavy coffee consumption (Djordjevic N et al 2008, Djordjevic N et al 2010).

There are some conflicts in the literature regarding the designation of this haplotype (Ingelman-Sundberg M et al 2007). The variant that defines this haplotype, CYP1A2: C-163A (rs762551), has different frequencies in different populations and the assignment of major and minor alleles therefore varies. Most publications have listed *1 F as C>A (Ingelman-Sundberg M et al 2007), but others have A>C (Cornelis MC et al 2006).

CYP1A2*1C

The only single-nucleotide polymorphism (SNP) for this haplotype is G-

3860A (rs2069514) has been well-defined and also seen in the literature as G-

2964A (Obase Y et al 2003). This haplotype is associated with decreased gene expression.

CYP1A2*1K

There are three variants that define this haplotype: T-739G (rs2069526), C-

729T (rs12720461) and C-1 63A (rs762551). This allele was shown firstly in

Ethiopians at a frequency of 3% (n = 173), in Spaniards at a frequency of 0.5% (n =

117), and in Saudi Arabians at 3.6% (n = 136) (Aklillu E et al 2003). CYP1A2*1K

allele has significantly reduced CYP1A2 enzyme activity in non-smokers compared

with *1A or *1F, using caffeine as a probe substrate (Aklillu E et aI 2003). 27 This haplotype has not been reported in Japanese (n = 350) (Takata K et al 2006) and Koreans (n = 50) and occurs at a very low frequency in Swedes (n = 193, 0.3%) (Ghotbi R et al 2007).

CYP1A2*3

There are two variants that define this haplotype 21 16G>A and 5347T>C (rs56276455). This haplo type is associated with decreased enzyme activity

(Chevalier D et al 2001, Zhou H et al 2004).

CYP1A2*4

There is one SNP for this haplotype 2499A>T (rs72547516). This haplotype is associated with decreased enzyme activity (Chevalier D et al 2001 , Zhou H et al

2004).

CYP1A2*6

There is one SNP for this haplotype 5090C>T (rs28399424). This haplotype is associated with decreased enzyme activity (Chevalier D et al 2001 , Zhou H et al

2004).

1.3.1.1.6 CYP1A2 Alleles Nomenc latu reNariant Annotations

Table 3: CYP1A2 alleles, their references SNPs and catalytic activity based on the Human Cytochrome P450 Allele Nomenclature Committee

CYP1A2 Location Nucleotide Change(s) and rs # Amino Acid Enzyme

Allele Change(s) activity

*1A Reference Reference Normal

*1C 5'-flanking region G-3860A (rs2069514) Gua2964Ade Decreased

*1 F Intron between the C-163A (rs762551). There is lIe386Phe Higher

noncoding exon 1 and confusion about the definition of the inducibility

exon 2 CYP1A2*1 F allele in the literature.

*1 K Intron 1 region C-729T (rs12720461) Cyt873Thy Decreased

*3 Exon 4 2116G>A (rs56276455) Asp348Asn Decreased expression 28

*4 Exon 5 2499A>T (rs72547516) IIe386Phe Decreased

expression *6 Exon 7 5090C> T (rs28399424) Arg431Trp Abolished

expression

1.3.1.1.7 CYP1A2 Genotype/Phenotype Relationship

Caffeine is a methylxanthine naturally available in some beverages and also considered as a pharmacological agent. Its main pharmacological effect is as a central nervous system stimulant. Caffeine is naturally occurring xanthine derivative like theobromine and the bronchodilator theophylline. Caffeine biotransformation occurs in several steps involving hepatic cytochrome P4501A2 (CYP1A2) (figure 7).

Caffeine is almost completely metabolized with 3% or less being excreted unchanged in urine (Begas E et al 2007, Marta K and Wadysawa DA 2008a).

The main route of metabolism in humans (70-80%) is via N-3 demethylation to paraxanthine (1,7 -dimethylxanthine or 17X) (Begas E et al 2007, Marta K and

Wadysawa DA 2008a and Benowitz N L et al 1995). This reaction is carried out by

CYP1A2 in the liver (Begas E et al 2007). Experimentally, human liver microsomes estimate that N-1 demethylation to theobromine (3,7-dimethylxanthine) and N-7 demethylation to theophylline (1 ,3-dimethylxanthine) accounts for approximately 7 to

8% of caffeine metabolism for each (Marta K and Wadysawa DA 2008b). The remaining 15% or less of caffeine undergoes C-8 hydroxylation to form 1,3,7-

trimethyluric acid (Figure 7) (Marta K and Wadysawa DA 2008b). 29

tH

CYP1,u

o H .. �& A-! :��N> " NJ- >-� I O� H �I

1�e'h nl • .,.,e·hylurc lOa

S-ace Ian no­ 5-i1o.l, Mono- &/oml I mn) 3-", hyur 6-1M1no-3-met'T)'luracd

Figure 7: Schematic representation of caffeine metabolites. CYP1A2 catalyzes the 3-demethylation of caffeine to paraxanthine and NAT2 catalyzes the acetylation of paraxanthin metabolites to AMFU

As discussed earlier, CYP1A2 is responsible for more than 95% of the primary metabolism of caffeine (Kalow W and Tang B K 1993). Therefore caffeine is used as a probe drug for CYP1A2 activity with the relative ratios of urinary metabolites used as an indicator of the flux through different parts of the pathway

(Begas E et al 2007). As well as paraxanthine, the major metabolites of caffeine in urine are 1 -methylxanthine (1 MX), 1-methyluric acid (1 MU), 5-acetylamino-6- formylamino-3-methyluracil (AFMU) and 1,7 -dimethyluric acid (17MU) (Begas E et al

2007).

Based on relative ratios of urinary metabolites, the ratio of paraxanthine/caffeine (17MXl137MX) has been measured from plasma after 4 hours of caffeine consumption to compare CYP1A2 genetiC polymorphism, enzyme activity 30 and the genotype-phenotype relationship in Sweden and Korean (Ghotbi R et al

2007).

The urinary molar ratio of (17MX + 17MU) / 137MX, taken at 4-5 h after caffeine ingestion, was identified from pharmacokinetic analyses as being better correlated for both NAT2 and CYP 1 A2 phenotyping (Butler MA et al 1992).

Evaluation of caffeine as an in vivo probe for CYP1A2 using measurements in plasma, saliva, and urine have been reported by Carrillo JA et al (2000) where

100 mg of oral caffeine has been consumed by subject to estimates CYP1A2 activity. They reported that both plasma and saliva total clearances of caffeine were highly correlated with each other (Carrillo JA et al 2000).

The ratio 17MXl137MX restricted to one sampling point taken 4 hours after dose, where highly correlated in plasma and saliva with the standard reference

(Carrillo JA et al 2000). Moreover, the ratio (AFMU + 1 MU + 1 MX + 17MU +

17MX)/1 37MX in a 0-24 hours urine sampling showed the highest correlation with the standard reference. (Carrillo JA et al 2000)

In conclusion they suggest the use of 17X1137X ratio for plasma or saliva, whereas (AFMU + 1 U + 1 X + 17U + 17X)/137X ratio is the recommended one for urine samples through a sampling interval of at least 8 hours, starting at time zero since caffeine intake (Carrillo JA et al 2000).

1.3.1.1.8 Clinical Impact, Drug Metabolism and Substrate Specificity of CYP1A2 Polymorphism

CYP1A2 is an important enzyme 'together with CYP1A1 and 1 B1 that

bioactivates a number of procarcinogens including polycyclic aromatic hydrocarbons

(e.g., benzo(a)pyrene), heterocyclic aromatic amines/amides (e.g. 2-amino-1-

methyl-6-phenylimidazo (4,5-b)pyridine), mycotoxins (e.g. aflatoxin B(1)) and some 31 natural compounds such as aristolochic acids present in several Chinese herbal medicines. This enzyme also metabolizes a lot of essential endogenous compounds

including retinols, melatonin, steroids, uroporphyrinogen and arachidonic acids.

(Wang B and Zhou SF 2009)

Several studies have indicated implications of CYP1A2 polymorph isms in cancer susceptibility, namely for pancreatic cancer (Suzuki H et al 2008), bladder

cancer (Altayli E et al 2009) and colorectal cancer (He X et al 2014).

Diseases associated with CYP1A2 include toxicity or absent response to

clozapine and porphyria cutanea tarda (Pruitt KD et al 2012).

Huge interindividual differences in the elimination of dr ugs that are

metabolized by CYP1A2 have been reported, and referred to both genetic mutations

and environmental factors (Ingelman-Sundberg M et al 2007, Zhou SF et al 2008,

Gunes A and Dahl ML 2008 and Zhou SF et al 2009b).

Xenobiotic metabolism is varied widely between individuals. Plasma levels of

a drug can vary more than 1000-fold between individuals upon the same drug

dosage (Ingelman-Sundberg M 2001 b, Cascorbi I 2006). These differences can

result from induction or inhibition and genetic polymorph isms of metabolizing

enzymes, or from physiological, pathophysiological and environmental factors

(Cascorbi I 2006). In fact, genetic factors are indicated to be responsible for 20-40%

of the differences between individuals in the response and susceptibility to drugs

and xenobiotics (Ingelman-Sundberg M 2001 a). Variability among individuals in drug

metabolism can result in decrease drug efficacies or undesirable toxic side effects.

There are wide interindividual differences (10- to 200-fold) in CYP1A2 (also ML called phenacetin O-deethylase) expression and activity (Gunes A and Dahl 1A2 mRNA 2008). Approximately 15- and 40-fold interindividual variations in CYP 32 and protein expression levels have been reported in human livers (Ikeya K et al

1989). These observations indicate a genetically measured difference in constitutive and/or inducible CYP1 A2 gene expression. Unimodal, bimodal, and trimodal distributions of CYP1A2 activity when determined by caffeine urinary metabolic ratios have been reported in different studies populations (Shimizu T et al 1991).

Pharmacogenetic studies on the impact of common CYP1A2 polymorphisms on drug clearance and response are still under investigation. More well-designed studies are important to explore the genotype-phenotype relationships of CYP1A2 in terms of drug clearance and response. Personalized pharmacotherapy and individualized dosing of drugs may require incorporation of both genetic and environmental factors.

Many clinical studies have been established to test the impact of CYP1A2 polymorphisms on drug clearance and drug response. For example, Obase Y et al

(2003) examined the effect of genetic polymorph isms in the 5'-flanking region to intron 1 of the CYP1A2 gene on theophylline metabolism in Japanese asthmatic patients (Obase Y et al 2003).

Among asthmatic patients, theophylline clearance was significantly lower in patients with the polymorphism at site G-2964A whose genotype was G/A

(intermediate) or AlA (slow) than in those whose genotype was G/G (rapid).

Therapeutic drug monitoring may be required in patients with the A allele at site

2964 in the CYP1A2 gene, due to low clearance level in those patients. Resistance

to clozapine therapy has been reported in smoking schizophrenic patients due to

low plasma drug levels harboring the 163 C/C (CYP1A2*1 F) genotype (Ozdemir V

et al 2001, Eap CB et al 2004).

Eap et al. (2004) recorded four smoking patients who did not respond to

clozapine therapy at usual dosage and they observed that those individuals were 33 ultra-rapid metabolizers of CYP1A2 carrying the *1 F allele (Eap CB et al 2004). On the other hand, higher plasma concentrations of clozapine and its metabolite N­ desmethylclozapine have been found in patients carrying two CYP1A2 variants associated with reduced enzyme activity (-3860A, -2467del, -163C, -739G, and/or -

729T) compared with those with one or none (Melkersson KI et al 2007).

Subjects with C-163C or C-163A genotype had lower plasma melatonin concentrations after administration of 6 mg melatonin compared with A-1 63A carriers (Hartter S, et al 2006). Another study in patients with rheumatoid arthritis

(n = 105) found that the *IF haplotype (C-1 63A) affected the risk for leflunomide­ induced organ toxicities (Bohanec GP et al 2008). Patients with A-1 63A genotype had a 9.7-fold higher risk for overall leflunomide-induced toxicity compared with patients with the CYP1A2*1F CIA or C/C genotype (Bohanec GP et aI 2008).

Porphyria cutanea tarda (PCT) is considered to be genetically predisposed to development of clinically overt disease through mutations and polymorphisms in genes associated with known precipitating factors. Christiansen et al. (2000) elucidated that the frequency of the highly inducible C/C (rapid) genotype at site C-

163A was increased in both familial and sporadic PCT. The authors presented that the C/C genotype was a susceptible factor for PCT (Christiansen et al 2000).

Paolini M et al. (1999) demonstrated that corresponding high levels of CYPs

in humans would predispose an individual to cancer risk from the widely bioactivated tobacco-smoke procarcinogens. No association has been reported between

CYP1A2 activity and sex and age based on Bebia Z et al (2004) in bioequivalence

study when they determined to which extent age and sex influence CYP

activity (Bebia Z et al 2004).

Acetaminophen as an effective analgesic and antipyretic agent, but causes

serious hepatic and renal toxicity at high doses by conversion of acetaminophen 34 to the toxic intermedia te N-acetyl-p-benzoquinone imine (NAPQI) through CYP1A2, CYP2 and E1, CYP3A4 (Sarich T et al 1997). In addition, using CYP1A2 enzyme inhibitors, it may increase risk of toxicity of lidocaine due to clearance inhibition

(Olkkola KT et al 2005).

The correlation was reported between caffeine breath test (CBT) results and tacrine blood levels as the first evidence supporting a critical role for CYP1A2 activity in the disposition of this drug in vivo (Fontana RJ et al 1996).

Mexiletine is mainly catalyzed by CYP2D6 and partially catalyzed by

CYP1A2 (Kusumoto Met al 2001). Xenobiotics inducing CYP1A1 mRNA expression have been found to induce CYP1A2 and CYP181, varied only based on the level and time course of induction (Krusekopf S et al 2003).

Generally, the 15q24.1 locus, including CYP1A2, is associated with blood pressure (BP). Higher CYP1A2 activity on the basis of positivity of the CYP1A2*1 F allele was linearly associated with lower BP after quitting smoking, but not while smoking. In non-smokers, the CYP1A2 variants were associated with higher reported caffeine intake, which in turn was associated with lower BP (Guessous I et aI 2012).

Some studies have also used caffeine as a modulator of disease etiology, looking at intake of caffeine containing fo ods and beverages with respect to disease risk. Two independent genome-wide association studies have examined the effect of variants on caffeine intake. They found associated variants in the regulatory region of CYP1A2 (rs2472304 and rs2472297) and in its regulator AhR (rs4410790 and rs6968865) (Cornelis MC et al 201 1, Sulem P et al 201 1).

CYP1A2 has been found to be essential for dosing of several anti psychotics

1A2 is the and for assessing both drug efficacy and adverse drug reactions. CYP 35 main CYP isoform responsible for clozapine metabolism (Eap CB at el 2004). Case studies presented ultrarapid metabolizers of clozapine that showed resistant to treatment. Increased clozapine doses and co-administration with the CYP1A2 inhibitor fluvoxamine were considered an alternative way to improve patient outcomes (Ozdemir Vet al 2001, Eap CB et al 2004).

CYP1A2 also alters the antithrombotic drug clopidogrel (Wang Z et al 2015).

Smoking elevates clopidogrel-mediated platelet inhibition (Bliden KP et al 2008,

Gremmel T et al 2009). This is related to induction of CYP1A2, which can play a significant role in colpidogrel activiation.

Like many of other CYPs, CYP1A2 is subjected to induction and inhibition by a number of compounds. Particularly, antofloxacin, carbamazepine, dihydralazine, furafylline, isoniazid, rofecoxib, clorgyline, thiabendazole, and are mechanism-based inhibitors of CYP1 A2. Reversible and irreversible inhibition of

CYP1A2 that may give an illustration of some clinical drug-drug interactions.

The process of drug interactions is a complex process because the medication interacts with one substrate of a specific cytochrome P450 pathway, does not mean it influences all substrates of that isozyme. Genetics, age, nutrition, stress, liver disease, hormones, and other endogenous chemicals also influence drug metabolism.

Substrate Specificity of Human CYP1A2

Phenacetin O-deethylation, caffeine 3-demethylation and ethoxyresorufin 0- deethylation are a commonly used marker reactions for CYP1A2. Particularly,

phenacetin O-deethylation is the most common marker reaction for CYP1A2 activity

in the in vitro study. (Yuan R et al 2002). 36 To a less extent, tacrine (1 ,2,3.4-tetrahydro-9-aminoacridine, 1- hydroxylation), melatonin (6-hyd roxylation) and tizanidine (oxidation) are also used as probes for CYP1A2 in vivo. For in vitro studies, phenacetin (O-deethylation), 7- ethoxycoumarin (O-deethylation), and 7-ethoxyresorufin (O-deethylation) are also used probes for determining CYP1 A2 activity (Waxman OJ and Chang TK 2006).

1.3.2 N-acetyltransferase-2 (NA T2)

1.3.2.1 Gene Location, Structure and Expression

Almost 22 NAT-like genes have been reported in 14 different prokaryotic and eukaryotic species (Butcher NJ et al 2002). The genes invariably have an intronless coding sequence that encodes for a protein between 254 and 332 amino acids in length (Butcher NJ et al 2002).

1.3.2.1.1 N-acetyltransferase Gene Family

Human NA T1, NA T2 as well as NA TP1 (pseudogene) were localized to the short arm of chromosome 8, more specifically in region 8p22. Both NA T1 and NA T2 genes show pronounced allelic variation (Hickman 0 et al 1994, Blume M et al 1990,

Butcher NJ et al 2002, Stanley LA and Sim E 2008, Hein OW 2009). The NAT loci are segregated by 170-360 kb only and are in the orientation

NA T1 -? NA TP 1 -? NA T2, with NA T1 being on the centromeric side of marker

08S261 and NA T2 coincid ing with marker 08S21 (Figure 8) (Matas Net aI 1997). 37

·0' pot 5.c: � � pb ------• J ;tJ : ... � -·-- .I�I ��------+-���---

E=:: _ pch" ..: ret · .� ')1 .\".-ffl - "';·3 -.-1 -u;, �. II / H ••. I .. 0>.-\ \ \ :'�:>1 �. >C :>1 :_A><: Lv>.-\.

Figure 8: Chromosomal location and structure of N-Acetyltransferase (NA T2) gene

The two functional NAT genes possess an 87.5% nucleotide identity, which reach up to 81 % homology at the amino acid level and around 80% with the corresponding sequence in NA TP (Blume M et al 1990). The whole transcript of

NA T1 is derived from a single exon; whereas NA T2 is derived from the protein encoding exon together with a second noncoding exon of 100 bp located about 8 kb upstream of the translation start site (Blume M et al 1990, Ebisawa T and Oeguchi

1991 ).

1.3.2.1.2 Reaction Mechanism, Structural Features, Variation and Functional Relevance of NAT2

The reaction is composed of two steps. Initially, acetyl coenzyme A binds to the enzyme and the acetyl moiety is shifted from the cofactor to a cysteine (Cys68 for the human isoforms) of the protein. The second step occurs when the binding of substrate to the acetylated enzyme where the acetyl moiety is transferred to the 38 substrate. Finally, the ac etylated product is separated from the enzyme (see aforementioned Figure 4) (Minchin RF et al 1992, Oelomenie et al 2001).

Sinclair (2000) reported the first crystal structure of an arylamine N­

acetyltransferase. A cysteine-histidine-aspartate catalytic triad was recorded in the

N-terminus of the protein (Sinclair JC 2000). In this regard, the protein has been divided into three domains. The first consists of a helical bundle, located from amino acid 1 to approximately 90, which forms one side of a cleft in which the cysteine involved in acetyl transfer resides.

The second domain consists of residues from approximately 90 to 210 amino acids and is located on the other side of the cleft. It mostly consists of l3-sheet structures. The last domain at the carboxyl terminus is a combination of l3-sheets and a-helices, which represents the biggest variation between species (Sinclair JC

2000).

Nine missense mutations where the alteration is in an amino acid (C-190T,

191 G>A, 341 T>C, 434A>C, 499G>A, 590G>A, 803A>G, 845A>C, and 857G>A),

and four silent where it occurs in the noncoding region (1 11T>C, 282C>T, 481C>T,

and 759C>T) have been recorded in different studies (Grant OM et al 1991, Vatsis

KP 1995, Hein OW et al 2000). The NA T2 gene has one non-coding exon around

8.6kb upstream of the intronless ORF (Figure 9) (Ebisawa T and Oeguchi T 1991,

Boukouvala S and Sim E 2005, Husain A et al 2007).

EXOtl 1 lntn>n 1 EXOt. 2

865"'1 bp _____ -11______

Figure 9: Structure of human NA T2 gene

Leff et al (1999) studied several different human NA T2 alleles in a yeast

expression system. They observed that three novel alleles, namely NA T2*50 39 haplotype (341 T>C), NA T2*14G haplotype (191 G>A, 282C>T, and 803A>G), and

NA T2 *60 haplotype (1 11C> T, 282C>T, and S90G>A), expressed proteins that had

N- and O-acetylation capacities similar to the expressed protein of the commonly occurring slow NA T2*58 allele, and significantly less than that of the wild-type

NA T2*4 allele.

The expression of NA T2 *58 and NA T2*50 haplotypes was reported to be significantly lower than that of the wild-type protein, suggesting that the base substitution at position 341, which is common to the NA T2*5 cluster, is sufficient for reduction in NA T2 protein expression. This was not observed to be the case for

NA T2*60 and NA T2*14G hap/otypes, which were expressed at levels comparable to wild-type. Contrarily, NA T2*60 and NA T2 *14G haplotypes were observed to be significantly less stable than the wild-type.

1.3.2.1.3 NAT2 Regulation

N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes for which three distinct enzymatic activities have been described. One is includes the acetyl coenzyme A (CoA) dependent N-acetylation of arylamines and arylhydrazines, a reaction usually associated with xenobiotic detoxification (Hein DW 2009).

The second one is also acetyl-CoA dependent and involves O-acetylation of

N-hydroxyarylamines (Hein DW 2009), typically generated through N-oxidation of arylamines by cytochrome P4S0 enzymes.

The third one is an acetyl-CoA independent N,O-acetyltransfer performed on

N-arylhydroxamic acids, generating highly reactive mutagenic compounds that bind to DNA. NATs have essential roles in the metabolism and detoxification of xenobiotics and therapeutic drugs, and are implicated in cancer risk because of their role in the activation or detoxification of carcinogens and their interaction with 40 environmental chemicals (Sim E et al 2008, Stanley LA and Sim E 2008, Sim E et al

2012).

1.3.2.1.4 NAT2 Variation and its Epidemiology

NA T2 is a polymorphic gene with almost 88 NA T2 alleles which have been assigned official symbols by the Arylamine N-acetyltransferase Gene Nomenclature

Committee, according to consensus guidelines (Hein DW et al 2008). NA T2*4 is the reference (or "wild type") allele for the respective gene, and the other variant alleles differ from this by one or more single nucleotide polymorphisms (SNPs).

The NA T2 gene has a high frequency of functional variation, which varies amongst populations that are ethnically different, and has high levels of haplotype variation (Patin E et al 2006b, Mortensen HM et al 201 1). SNPs within the NA T2 gene cause several changes in NAT2 function by altering enzyme stability, altering affinity for substrate, or a protein that is targeted for proteasome degradation (Walraven JM et al 2008, Hein DW 2009).

NA T2 genotypes have been classified into three different phenotypes: "slow acetylator" (two slow alleles), "intermediate acetylator" (1 slow and 1 rapid allele), and "rapid" acetylator (2 rapid alleles) (Stanley LA and Sim E 2008). Some papers

report rapid as any genotype containing NA T2*4 and slow as any non-carriers of NA T2*4 acetylators (Soejima M et al 2007). However, other rapid alleles that

have been fo und apart from NA T2 *4 are NA T2 *1 1 A, NA T2 *12A-C, NA T2 *13A,

NA T2 *1B. In addition, within the slow acetylator genotype group there is

heterogeneity in phenotype due to variation in enzyme activity conferred by different

alleles (Hein OW et al 1995, Cascorbi I et al 1999, Hein OW 2009, Ruiz JO et al

2012), which may affect the ability to identify significant associations (Selinski S et al

201 3b). 41 The frequency of the slow acetylator phenotype differs markedly among ethnic groups (Evans DAP 1984), and this is referred to the differing frequencies of the polymorphisms that correspond to the slow acetylator alleles. In Caucasian and

African populations, the frequency of the slow acetylation phenotype varies between

40 and 70%, whereas it ranges from 10 to 30% in Asian populations, such as

Japanese, Chinese, Korean, and Thai (Meyer UA and Zanger UM 1997). Caucasian and African populations have high frequencies of NA T2 *5 haplotypes (>28%) and low frequencies of NA T2*7 haplotypes « 5%), while Asian populations have low incidences of NA T2*5 haplotypes «7%) and higher incidences of NA T2*7 haplotypes (>10%). Moreover, NA T2*14 haplotypes are almost absent from

Caucasian and Asian populations «1%), but are present in African populations at comparably higher frequencies (>8%).

Several studies examining the variation of NA T2 haplotypes between different populations and ethnicities support the hypothesis suggesting the NAT2 slow acetylator phenotype was positively selected for the transition to an agricultural! pastoral lifestyle from a hunter-gatherer/ nomadic lifestyle, leading to changes in diet and thus exposure to different xenobiotics (Patin E et al

2006a, Magalon H et al 2008, Sabbagh A et al 2008, Luca F et al 2008).

For example, slow acetylator status is higher among Tajik populations

(agriculturists) compared to Kirghiz populations (nomads) in Central Asia (Magalon

H et al 2008), and a high frequency of rapid or intermediate status is observed in

hunter-gatherer populations in Western/ Southern Africa (Kung San, Bakola Pygmy,

Biaka Pygmy populations) (Patin E et al 2006b, Mortensen HM et al 201 1). In India,

the frequency of slow acetylators (based on genotype) was higher than rapid

acetylators in areas where a vegetarian diet dominates, and the opposite was found

in areas where non-vegetarian diet is more frequent (Khan N et al 201 3). 42 Yuliwulandari et al (2008) detec ted 23 different variants in the promoter and coding regions of the NA T2 gene among 212 Indonesian individuals and they found that phenotypes for rapid, intermediate and slow acetylators were 13.6%, 50.8%, and 35.6%, respectively. The frequency of slow acetylators was similar to that recorded for other Southeast Asian populations.

Magalon et al. (2008) genotyped 138 individuals from 6 populations in central Asia, they fo und that the Tajiks and Kazakhs showed the highest frequency of the slo w acetylator haplotype *58, ranging from 22 to 26% . The *6A haplotype was exhibit at high frequencies in the Tajik populations (39 to 45%), whereas it was at the lowest frequency in Kazakhs (13%). The Ka zakhs exhibited the highest frequency (23%) of *78, which is mainly restricted to East Eurasian populations. The

'rapid' haplotype *4 was observed at high frequency in the Kirghizs (46 to 48%) and in the Uzbeks (42%), but at nearly 2-fold lower frequencies in the Tajiks (23 to 26%), and intermediate frequency in the Kazakhs (38%). Overall, the Tajiks showed significantly higher proportions of slow acetylators (55% to 63%) as compared to the

Uzbeks, Kirghizs, and Kazakhs, where the proportions of slow acetylators ranging from 26 to 35% (Table 4 and 5) (Magalon H et al 2008).

Table 4: Frequency of important SNPs in human NA T2 gene (Guaoua S et a1 2014)

Ethnic groups NA T2*5 NA T2*6 NA T2 *7 NA T2 *14

Caucasians 0.46 0.285 0.029 0.00 Germans 0.425 0.278 0.013 0.001 Americans 0.437 0.266 0.019 0.001 Southern Korean 0.01 0.224 0.132 0.00 Spanish 0.47 0.25 0.006 0.004 Southern Brazil 0.289 0.104 0.02 1 0.014 Egyptian 0.497 0.26 0.028 Argentine 0.37 0.256 0.08 0.013 Omanis 0.44 0.27 0.04 0.00 Senegalese 0.322 0.188 0.00 0.084 Tunisians 0.315 0.175 0.15 0.05 South Africa 0.361 0.17 0.067 0.103 43 Japanese 0.019 0.23 0.01 1 Indians 0.33 0.38 0.03 Moroccans 0.53 0.25 0.02 0.04

Table 5: Frequency of other SNPs in human NA T2 gene (Luca F et al 2008)

Haplotype Dendi Amha Orom Egyptla Italian Gree Czee Mordvin RusSI Khanty Yakuts Chukeh ra ns s k 0 s hs an MansI ee 0 006 008 0. 12 024 021 "4 02 021 033 0.14 0.38 0.35 '5 0 0 0 04 0 01 0 0 01 o o 0.04 0 0.03 o 023 053 033 049 "58 032 038 039 050 0.33 029 019 019

*5C 0 05 0 0.04 0 01 0 03 0 04 006 o 0 0.07 o o '50 005 0 0 0 0 0 0.06 o 0 0 o o

*5G 005 003 0 0 0 0 o o 0 0 o o

*5J 0 0 0 0 0 0 004 o 0 0 o o

*6 027 027 021 027 034 0.29 0.26 0.11 0.08 0.11 0.25 0.27

*68 0.09 0 0 0 0 0.01 o o 0 0 o o

*6C 0 05 0 0 0 0 0 o o 0 0 o 004

'7 0 0 0 0 0 0.01 o o 0 0 o 0.04

?8 0 003 017 001 0.03 0.03 o 0.07 004 0.18 013 0.08

*11 0 0 0 0 0.01 0 o o 0 0.1 1 o o

*12 005 003 008 0.01 0 0 o o 008 0.04 o 0.04

*128 0 05 0 0 0 0 0 o o 0.04 0 o o

*12C 0 0 0 0 0 01 0 01 o 007 0 0.04 0.03 o

*13 0.14 003 004 007 0.01 001 o 0.04 0.04 0.04 o o

Important Variants

NA T2 alleles containing the 191 G>A (rs 1801279), 341 T>C (rs1801280),

590G>A (rs 1799930), and/or 857G>A (rs 1799931) missense substitutions are

associated with slow acetylator phenotype(s). Striking ethnic differences in the

frequencies of these missense substitutions (Grant OM et al 1997) play an important

role in ethnic differences in frequency of slow acetylator alleles (Cascorbi I et al

1999) and phenotype(s) (Weber WW and Hein OW 1985). For example, the

191 G>A substitution commonly represents the NA T2 *14 gene cluster that is

available in greater extent in African Americans and native Africans, but it is virtually

absent in Caucasian populations. 44

Lin et al. (1994) detected 4 mutations 191 G>A, 481 C>T, 590G>A, and

857G>A accounted for nearly all slow acetylator alleles among blacks, whites, Asian

Indians, Hispanics, Koreans, Japanese, Hong Kong Ch inese, Taiwanese, Filipinos, and Samoans.

Cascorbi et al. (1995) found 7 different alleles of the NA T2 gene coding for the 'slow' phenotype. They observed a slow acetylation genotype in 58.9% of the

844 unrelated German subjects studied (Cascorbi et al 1996). In vivo acetylation capacity of homozygous wildtype subjects was significantly higher than in heterozygous genotypes. All mutant alleles showed low in vivo acetylation capacities, and the term 'slow' genotypes differed significantly among each other, as reflected in lower acetylation capacity of some alleles as compared with others.

Other Important Variants

Other NA T2 alleles that have been reported to be associated with slow acetylator phenotype are those containing the A-434C (rs 72554616) and C-190T

(rs 1805158) (Oai Z et al 2008). However, till now there are no reports which detect

SNP rs72554616 influence on drug metabolism or any disease so it may not have any clinical impact. On the other hand, only one report of isoniazid side effect has been detected with SNP rs1805158 (Fuselli S et al 2007).

Other Variants

Other variants include G-499A (rs 72554617) which represent haplotype *10, shows slow catalytic activity but substrate dependent. Variant 803A>G (rs 1208)

presented in allele *12, variant 282C> T (rs 1041983) presented in allele *13 and

variant A-845C (rs 56054745) presented in allele *18 have shown to have rapid

catalytic activities that is also included in the metabolism of certain drugs like 1,7-

dimethylxanthine, antivirals for treatment of HIV infections, drugs for treatment of 45

tuberculosis, hydralazine, sulfamethoxazole and trimethoprim (Weber WW and Hein

OW 1985, Stanley LA and Sim E 2008, Sim E et al 2012, McDonagh EM et al 2014).

1.3.2.1.5 NAT2 Important Haplotypes

NA T2*4 is considered the wild-type allele because of the absence of any of these substitutions and based on many studies in many ethnic groups, NA T2*4 is not the most common allele (Hein OW et al 1994, Hein OW et al 1995, O'Neil WM et aI 2000).

NA T2* 5, NA T2 * 6, NA T2* 14, and NA T2* 7 alleles have low catalytic activity associated with the slow acetylator phenotype, whereas NA T2 *12 and NA T2 *13 alleles associated with high catalytic activities that show rapid acetylator phenotype similar to NA T2*4 (Hein OW 1994, Hein OW et al 1995, O'Neil WM et al 2000).

1.3.2.1.6 NAT2 Alleles Nomenclatu reNa riant Annotations

Since 1995 many alleles and their nomenclature have been identified and published (Vatsis KP et al 1995). The Human Gene Nomenclature Committee has agreed that the symbol NAT be assigned to the arylamine N-acetyltransferase genes. NA T2 alleles *1, *2 and *3, have been found but these are non-human NA T2 alleles. Based on this, human NA T2 alleles start with allele *4 (wild type) (table 6)

(Hein OW et al 2008).

Table 6: NA T2 alleles and its references SNPs and catalytic activity

NA T2 Allele Nucleotide Change(s) and rs # Amino Acid Phenotyped (Haplotype) Change(s) *4 Reference Reference Rapid *5A 341T>C (rs1801280) 11 14T Slow 48 1 C>T (rs 1 799929) L161 L (synonymous)

*58 341T>C (rs 1801280) 11 14T Slow 481 C>T (rs1 799929) L161 L 803A>G (rs1 208) (synonymous) K268R * Slow 5C 341T>C (rs1801280) 1114T 46

803A>G (rs1 208) K268R *6A 282C>T (rs1041983) Y94Y Slow 590G>A (rs1 799930) (synonymous) R197Q *68 590G>A (rs1 799930) R197Q Slow *6C 282C>T (rs 1 04 1 983) Y94Y Slow 590G>A (rs1 799930) (synonymous) 803A>G (rs 1208) R197Q K268R ' 7A 857G>A (rs1 799931) G286E Slow *78 282C>T (rs1041983) Y94Y Slow 857G>A (rs1 799931) (synonymous) G286E *7e 282C>T (rs1041983) Y94Y 803A>G (rs1 208) (synonymous) 857G>A (rs 1799931) K268R G286E *14A 191G>A (rs1801 279) R64Q Slow *148 191 G>A (rs1801 279) R64Q Slow 282C>T (rs1041983) Y94Y (synonymous) *14C 191 G>A (rs1801279) R64Q Slow 34 1T>C (rs1801280) 1114T 481 C>T (rs 1 799929) L161L 803A>G (rs1 208) (synonymous) K268R

1.3.2.1.7 NA T2 Genotype/Phenotype Relationship

The caffeine metabolite Paraxanthine can undergo acetylation by NAT2 to form 5-acetylamino-6-formylamino-3-methyluracil (AFMU) (Thorn CF et al 2012).

Caffeine can be utilized as a non-toxic probe drug in vivo for examining the

acetylation phenotype status; by determining metabolite ratio AFMU/1 -methyl

xanthine (1 MX) in urine after caffeine consumption, a bi- or tri-modal pattern in a

given population is determined (Grant OM et al 1984, Grant OM et al

1990, Kaufmann GR et al 1996). AFMU/AFMU+1X+1-methyluric acid (1 U),

AFMU+5-acetylamino-6-amino-3-methyluracil (AAMU)/AFMU+AAMU+1 MX+1 MU or

AAMU / AAMU+1X+1U metabolite ratios can also be used to measure acetylation

phenotype status (Jetter A et al 2004, Begas E et al 2007, Ojordjevic N et al 201 1,

Ojordjevic N et al 2012).

Therefore, most commonly used probe drug for NAT2 phenotype

measurement is caffeine (Grant OM et al 1984, Lorenzo B and Reidenberg MM 47

1989, Leyland-Jones B et al 1999) and excellent NA T2 genotype/phenotype correlations have been recorded (Cascorbi I et al 1995, Grant OM et al 1997).

Although administration of caffeine is a relatively noninvasive method for measuring

NAT2 acetylator phenotype, the method used involves determination of the quantity of other metabolites. Thus, genetic and environmental factors may influence the levels of the metabolites used in the phenotype determination. For example, NAT1 phenotype affects the urinary caffeine ratio utilized for determination of NAT2 phenotype (Cribb AE et al 1996).

No association has been reported between NAT2 acetylation activity and sex, race, age, education, smoking , physical activity, weight, vitamin E supplements, alcohol and dietary intakes of red meat, processed meat or cruciferous vegetables or use of menopausal estrogens (Le Marchand I et al 1996).

In one study, up to 54% of the differences in acetylation activity measured by caffeine test could be demonstrated by NA T2 genotype (homozygous wildtype, homozygous variant or heterozygous determined by PCR-RFLP), although it was found that use of phenotyping had some advantages in sensitity over genotyping (Le

Marchand Let al 1996).

The original method for determining the NAT2 genotype used Southern blot analysis of genomic DNA from leukocytes but soon afterwards, a PCR-based RFLP

method was described (Mashimo M et a1 1992, Abe M et al 1993). In addition, use of

the urinary molar ratio of AFMU/1-methylxanthin to determ ine NAT2 phenotype it

was described and validated at around the same time (Butler MA et al 1992). 48

1.3.2.1 .8 Clinical Impact, Drug Metabolism and Substrate Specificity of NAT2 Polymorphism

The association between acetylator status and the risk of various diseases has been extensively recorded, and reviewed in detail (Evans DAP 1984, Clark OW

1985, Evans DAP 1993).

Altered risk with either the slow or rapid phenotype has been reported for several diseases including bladder, colon and breast cancer, systemic lupus erythematosis, diabetes, Gilbert's disease, Parkinson's disease (PO) and

Alzheimer's disease. Humans are exposed to many toxic NAT substrates including the food-derived heterocyclics present in the diet as well as arylamines such as 4- aminobiphenyl and l3-naphthylamine present in tobacco smoke (Nagao M et al

1996, Smith CJ et al 1997, Besarati NA et al 2000, Sram RJ and Binkova B 2000).

Their influences cause several adverse effects with a lot of drugs like dipyrone, drugs for treatment of tuberculosis, hydralazine, isoniazid, pyrazinamide, sulfamethoxazole, sulfapyridine, sulfasalazine and caffeine (Weber WW and Hein

OW 1985, Stanley LA and Sim E 2008, Sim E et al 2012, McDonagh EM et al 2014).

In cancer, NAT2 phenotype/genotype may be a risk factor. NAT2 has been implicated in chemical carcinogenesis pathways. Unlike the relatively rare but highly penetrant genes included in familial cancers, those genes responsible for metabolic polymorphisms have low penetrance and cause only a moderate increase in cancer

risk. Nevertheless, their widespread occurrence in the general population suggests that they are a significant contributor to individual risk (Weber WW and Hein OW

1985, Sim E et al 2008, Stanley LA and Sim E 2008, Sim E et al 2012, McDonagh

EM et al 2014).

For that, polymorphisms in the NA T2 genes have been examined for an

association with cancer risk, however the findings are inconsistent; this may be due 49 to the complex nature of cancer etiology and the multiple factors that contribute to susceptibility (Weber WW and Hein OW 1985, Sim E et al 2008, Stanley LA and Sim

E 2008, Sim E et al 2012, McDonagh EM et aI 2014).

In 1986, Lang et a/ first illustrated an association between the slow acetylator phenotype and bladder cancer while the rapid acetylator phenotype was suggested to have an increased risk factor for colon cancer (Lang et al 1986, lIett et al 1987,

Wohlleb JC et al 1990). Other studies have been unable to confirm these findings

(Welfare MR et al 1997, Chen J et al 1998, Brockton N et al 2000).

Cartwright et al (1982) divided the ratio that used to measure the phenotype status into 4 groups: 0.3 and greater correspond to rapid acetylators, and the other three (0.01-0.09; 0.1-0.19; and 0.2-0.29) correspond to different levels of slow acetylator phenotype. Urinary bladder cancer risk is markedly elevated in NAT2 slow acetylator phenotype, particularly with the slowest NAT2 phenotype (Cartwright RA et al 1982). This same conclusion has been reported by different studies including some recent large genome-wid association studies (Brockmoller J et al 1996,

Okkels H et al 1997, Filiadis IF et al 1999, Hamdy SI et al 2003, Lubin JH et al 2007,

Garcia-Closas M et al 201 1, Figueroa JD et al 2014).

Another study showed an enhanced effect for smoking intensity and bladder cancer in NAT2 slow acetylators which increases with intensity (Lubin JH et al

2007).

Several studies have been reported that rapid N-acetylation phenotype was

associated with an increased risk of colorectal cancer, particularly, in patients with

right-sided cancer. However, the frequency of the *7A haplotype associated with the

slow acetylator status, was increased among colorectal cancer patients on the left

side and along the sigmoid/rectal region (Lang NP et al 1986, lIett KF et al 1987,

Lee EJD et al 1998). 50

Women with very slow acetylator genotype (homozygous for NA T2*5) who smoked for 20 years showed an increased breast cancer risk when they were compared to individuals with rapid NA T2 genotype (Vander HL et al 2003).

Some other studies sho wed no association between NAT2 acetylator phenotype and breast cancer (Webster OJ et al 1989, Ladero JM et al 1991).

Contrarily, rapid acetylator phenotype was associated with breast cancer risk in other studies (Bulovskaya LN et al 1978, Philip PA et al 1987, Sardas S et al 1990).

In yet other studies, the association between NA T2 acetylator genotype and breast cancer has been examined in relation to diet. It was observedtha t red meat consumption and NA T2 genotype were not associated with breast cancer risk

(Ambrosone CB et al 1998, Gertig OM et aI 1999). However, in another study, rapid

/ intermediate NA T2 genotypes were associated with breast cancer risk in women who consistently consume very well-done meat (Deitz AC et al 1999).

The correlation between NA T2 genotype and breast cancer among smoking women has produced inconsistent results (Ambrosone CB et al 1996, Hunter OJ et al 1997, Millikan RC et al 1998, Huang CS et al 1999). Mixed results were observed for NA T2 genotype and risk of breast cancer (Zgheib NK et al 2013, Fernandes MR et al 2013) and also oesophageal cancer (Jain M et al 2007, Malik MA et al 2009,

Wang Let al 2014).

The studies that investigate the risk association between NA T2pheno type

and the susceptibility to lung cancer were either negative (Burgess EJ et al 1985) or

showed a slight overrepresentation of rapid acetylators (Roots I et al 1988, Philip PA

et al 1988, Martinez C et al 1995, Hirvonen A et al 1995, Cascorbi I et al 1996,

Hirvonen A et al 1996, Nyberg F et al 1998, Bouchardy C et al 1998). 51 Rapid NAT1 and/or NAT2 acetylators activate N-hydroxy-heterocyclic amine carcinogens within the colon to their ultimate carcinogenic forms, thereby predisposing them to colorectal cancer (Kirlin WG et al 1991, Turesky RJ et al 1991 ,

Bell OA et al 1995). Several studies have observed an association between rapid

NAT2 acetylator phenotype and colorectal cancer (Lang NP et al 1964, Ilett KF et al

1987, Gil JP and Lechner MC 1998), whereas other studies did not (Ladero JM et al

1991, Rodriguez JW et al 1993, Shibuta K et al 1994, Probst-Hensch NM et al 1995,

Spurr NK et al 1995, Hubbard AL et al 1997). Some studies have recorded an association between rapid NAT2 acetylator phenotype and colorectal cancer for

Individuals consuming well-done meat and, presumably, higher levels of heterocyclic amine carcinogens (Lang NP et al 1994, Skog K et al 1995, Roberts-Thomson IC et al 1996, Welfare MR et al 1997, Chen J et al 1998, Kampman E et al 1999).

Consistent with that, rapid NAT2 acetylators who consumed pan-fried meats had higher levels of urinary mutagenicity than slow acetylators (Marini OM et al

1997). Whereas it is only limited to homozygous rapid (*41*4) acetylators (Gil JP and

Lechner MC 1998), this finding was also observed for lung (Cascorbi I et al 1996) and laryngeal (Henning S et al 1999) cancers. Bell OA et al 1995 recorded an association between the NA T1*10 allele and colorectal cancer, and the risk were highest among NAT2 rapid acetylators. Another study also found a higher risk for colorectal cancer in individuals who consumed well-done meat and possessed both the NA T1 *10 allele and rapid acetylator NA T2 genotype (Chen J et al 1998).

In another study, the homozygous slow acetylator NA T2 genotype was correlated with an increased risk of oral/pharyngeal cancer, but not laryngeal cancer

(Jourenkova-Mironova N et al 1999). However, the homozygous rapid acetylator

(*41*4) genotype was strongly correlated with laryngeal cancer in a German study

(Smelt VA et al 1998). 52 Several studies conducted to examine the associations between acetylator genotypes and prostate cancer found no relationship between NA T2 genotype and prostate cancer (Agundez JAG et al 1998, Wadelius M et al 1999). Additionally, aromatic amine N-acetyltransferase activity levels in human prostates were independent of NA T2 genotype (Agundez JAG et al 1998).

Head and neck cancers are strongly correlated with smoking, and several studies have investigated the role of NA T1 and NA T2 polymorph isms and the risk of head and neck cancer in smokers. The slow NAT2 acetylator phenotype was observed to be associated with the development of head and neck cancer in

Caucasians (Or6zdz M et al 1987, Gonzalez MV et al 1998) and with the development of oesophageal cancer in Japanese (Morita S et al 1998).

Another study showed that NA T2 slow acetylator genotype can be a small, low penetrance risk factor for head and neck cancer (Zhang L et al 2014). Seeking further studies is important to have clear picture about the associations between NA T2 variants and susceptibility to other cancers (Boukouvala S and Fakis

G 2005, Sim E et al 2008, Butcher NJ and Minchin RF 2012).

Chan et al. (2003) found that the frequency of the slow acetylator genotype for NA T2 was significantly higher in Parkinson's disease (PO) patients than in a control group after adjusting age, sex, and smoking history in Hong Kong Chinese.

A significant associations with PO for NAT2 was also reported in a meta analysis

(Tan EK et al 2000). Similarly, Bandmann 0 et al (2000) stated that "the slow phenotype appears to increase risk of PO".

Increased risk of the late-onset Alzheimer's disease has been observed with

the rapid NAT2 phenotype in non-a poE epsilon 4 carriers (Ogawa M 1999). 53 NA T2 polymor ph isms and acetylator phenotype may affect risk of other complex mult ifactorial diseases (including asthma and diabetes), however, the results are conflicting and furtherstudies are needed (Ladero JM 2008, Batra J and Ghosh B 2008).

The development of molecular epidemiological studies exploring the

relationship between the acetylation polymorphisms and cancer are still incomplete

but there has been considerable progress recently due to the availability of genome- wide association studies (Chung CC et al 201 0). The inconsistency in the human

epidemiological studies may be related to differences in carcinogen exposures,

genotype and/or phenotype methods, insufficient sample sizes, and/or other

susceptibility genes and factors.

The drug adverse effects related to NAT2 phenotype mostly occurred as the

result of a shift in the metabolic pathways responsible for the activation and

detoxification of the drug. This is best demon strated by the amine-containing

sulphonamides, such as sulphamethoxazole, that undergoes hydroxylation to a

reactive N-hydroxy metabolite capable of covalently binding to macromolecules and

giving rise to idiosyncratic adverse reactions (Figure 10) (Spielberg SP 1996).

Hydroxyl.don •

Covalendy bind to Macromolecules leading to Idlosyne de Adverse Reaction

ation and detoxification Figure 10: The metabolic pathways responsible for the activ of the drug 54 These drugs can also be acetylated by NAT2 to non-reactive N-acetyl metabolites. In slow acetylators, a higher proportion of the drug is N-hydroxylated and based on that individuals with slow acetylation status are at a greater risk of sulphonamide-induced toxicity (Das KM and Dubain R 1976, Shear NH et al 1986,

Cribb AE et al 1996).

The major focus of NAT2 pharmacogenetic studies are on the association of anti-tuberculosis (anti-TB) drugs and drug-induced hepatotoxicity, liver injury (Dill), or hepatitis.

Sulfamethoxazole is acetylated to N-acetylsulfamethoxazole, or oxidized to sulfamethoxazole hydroxylamine (a reactive metabolite that is responsible in toxicity) by CYP450 enzymes (Davis CM and Shearer WT 2008). The association between NA T2 genotypes and sulfamethoxazole pharmacokinetics (PK) have also been supported in renal transplant patients treated with an immunosuppressive regimen, significantly higher sulfamethoxazole concentrations in slow acetylators

(defined as homozygotes or compound heterozygotes for *5, *6, or *7 variants) are seen compared to rapid acetylators (homozygous *41*4) (Kagaya H et al 2012).

NAT2 is important pathway in the metabolism of isoniazid (INH), mediating its biotransformation to the metabolite acetyl-INH, which is hydrolyzed to isonicotinic acid or acetyl-hydrazine and subsequently to the non-toxic diacetylhydrazine, or

hydrolyzed to hydrazine (Preziosi P 2007, Tostmann A et al 2008, Metushi IG et al

201 1, Mahapatra S et al 2012, Daly AK and Day CP 2012). Liver toxicity of INH

treatment derives from INH itself (a hydrazine derivative) and its metabolites,

including acetyl-hydrazine, hydrazine and ammonia, and is thought to include the

formation of reactive oxygen species that can cause necrosis and autoimmunity

(Mitchell JR et al 1975, Preziosi P 2007, Tostmann A et al 2008, Fukino K et al 55 2008, Metushi IG et al 201 1) and may also involve epigenetic effects (Murata K et al 2007).

Consequ ently, NAT2 slow acetylators have been associated with an increas ed risk of hepatotoxicity / liver injury / hepatitis induced by anti-TB drug treatment when compared to rapid acetylators (Bose PO et al 201 1, Chamorro JG et al 2013 and Gupta VH et al 201 3).

NA T2 variants 590G>A and 857G>A were independently observed at a significantly higher frequency in children with co-trimoxazole induced adverse drug reactions (AORs) compared to those without (ZieliriskaE et al 1998).

In another study on Japanese patients who were treated with co-trimoxazole, it was found to have higher risk of adverse events with slow acetylator status

(determined in this study by NA T2 genotypes *6A/*6A, *6A1*78, *781*78) compared to rapid acetylators (genotypes *41*4, *41*58, *4/*5E, *4/*6A, *41*78) (Soejima M et aI 2007).

The increased risk of developing side effects such as neurotoxicity or haemolytic anaemia, to dapsone therapy is very similar to that elucidated for the sulphonamides (May OG et al 1990). The most severe incidence of toxicity found in individuals with a slow acetylator phenotype who are rapid hydroxylators (Bluhm et al 1999).

While slow acetylators are at a greater risk of toxicity from sulphonamides and dapsone, amonafide which is drug used for treatment of various cancers it is an arylamine that show elevated incidence of adverse reactions in rapid acetylators The drug after being given standard dose compared to the slow acetylators.

systemic toxicity undergoes N-acetylation to an active metabolite that contributes to two groups were (myelosuppression). Based on that, different doses for the 56 recommended. However, the drug displayed variable and unpredictable toxic effects failed to re ach phase III clinical trial primary end points (Ratain MJ et a1 1991, 1993,

1995, 1996, Innocenti F et al 2001, Freeman CL et al 2012).

One study found a reduction in the antihypertensive activity of hydralazine in the rapid acetylators which required a 40% higher dose to show similar therapeutic effect compared with slow acetylators. This variation appeared to be due to alteration in the bioav ailability of the drug which reduced from 33% in slow acetylators to less than 10% in rapid acetylators (shepherd AM et al 1980).

Studies revealed that rapid acetylators show lower hydralazine plasma concentrations and area under the concentration-time curve compared to slow acetylators (Israili ZH and Dayton PG 1977, Weber WW and Hein OW

1985, Gonzalez-Fierro A et al 201 1, Spinasse LB et al 2014).

Sulfasalazine consists of sulfapyridine that metabolizes to N-acetyl­ sulfapyridine which is significantly diminished in slo w acetylators (carriers of two variant alleles NA T2*58, NA T2*6A, NA T2*78 or NA T2 *5, NA T2*6 and NA T2 *7) compared to both intermediate (one variant and one NA T2 *4 haplotype) and rapid acetylators (NA T2*41*4)(Yamasaki Yet al 2008, Ma JJ et al 2009). Slow acetylators have higher concentrations and elimination half-life of sulfapyridine (based on genotyping NA T2 SNPs rs1041983, rs1801280, rs1 799929, rs1 799930 and rs1 799931) (Kuhn UD et al 2010).

A supported prospective study of female rheumatoid arthritis (RA) patients treated with sulfasalazine detected 4 patients who had reported adverse effects to

sulfasalazine in a one year period - none had the NA T2*4 haplotype, each carrying two variant alleles in Japan (Soejima M et al 2008). 57

In summary, the examples discussed above have highlighted the essentials of NA T2 as a genetic influence on drug pharmacokinetics and pharmacodynamics

(Table 7) (Ratain MJ et al 1996, Innocenti F et al 2001).

Table 7: Effect of acetylator status on drug response and toxicity (Butcher NJ et al 2002)

Drug Phenotype Effect Dapsone Slow Neurotoxicity Sulphamethoxazole Slow Hypersensitivity Hydralazine Slow Systemic lupus erythematosus Rapid Decreased therapeutic effect Isoniazid Slow Interaction with phenytoin Interaction with rifampicin Cotrimoxazole Slow Various adverse reactions Sulphasalazine Slow Various toxicities Hepatotoxicity. nausea /vomiting Amonafide Rapid Leukopenia Procainamide Slow Systemic lupus erythematosus Phenelzine Rapid Decrease therapeutic effect

Some studies demonstrated that bacterial NAT from the gut flora has a role in the activation and detoxification of xenobiotics in the host organism and may play an essential role in the metabolism of anti-inflammatory drugs, such as 5- aminosalicylic acid (Trepanier LA et al 1997, Delomenie C 2001).

Substrate Specificity of Human NAT2

There is no specific substrate structure that is uniform for the different isoforms of NAT, although, p-aminobenzoic acid (PABA), p-aminobenzoyl glutamate and p-aminosalicylic acid (PAS) are identified as specific substrates for human

NA T1. These substrates can be featured by the availability of relatively small

hydrophilic substitutions in the para position of the aromatic ring. On the other hand,

sulfamethazine, procainamide and dapsone are acetylated primarily by human

NA T2. Other compounds such as 2-aminofluorene are excellent substrates for both

human NA T1 and NA T2 (Minchin RF et al 1992, Delomenie C et al 2001). 58

Endogenous substrates for human NA T2 are not well known, although, in rodents N-acetyltransferases metabolize a number of aromatic and heterocyclic amine carcinogens that form tumors (Layton OW et al 1995). Aromatic amines and hydrazines (N-acetylation) and N-hydroxy-aromatic and heterocyclic amines (0- acetylation) are both examples of acceptor substrates that are deactivated (N­ acetylation) or activated (O-acetylation) by NAT2 (Hein OW 1988).

1.4 Potential Contribution and Limitation of the Study

Time constraints have been the main challenge that we have faced during this study. Molecular and epidemiological research involving human subjects requires obtaining the appropriate ethical approvals which involved numerous communications with managers and administrators in several institutions. This was an absolute pre-request before sample collection.

Moreover, our study required volunteers who had to drink caffeinated drinks and come back after 2 hours for sample collection which was inconvenient for some individuals who refused to participate. Additionally, not all the places were considered appropriate for sample collection which limited the number of centers that we could use for subjects recruitment.

Interestingly, the level of consanguinity in the UAE varies with more than

50% of marriage are consanguineous. This clearly may influences the genotypes frequencies and may result in their deviation from Hardy-Weinberg. 59

Chapter 2: Aims and Objectives

2.1 Aims

This study has been conducted to determine CYP1A2 and NA T2 genotypes and their frequencies and correlations with the corresponding phenotypes in

Emiratis.

2.2 Specific Objectives

./ Literature evaluation of CYP1A2 and NA T2 polymorphism and its association

with diseases, drug response and toxicity .

./ Recruitment of a sample of volunteers that is genetically representative of

Emiratis and willing to go through phenotyping and genotyping analysis .

./ Measurement of CYP1 A2 phenotyping status of the recruited samples .

./ Determining the polymorphic CYP1 A2 alleles and genotype frequencies of the

recruited samples and examine if they follow or deviate from the Hardy­

Weinberg equilibrium .

./ Defining the correlation between CYP1A2 genotyping and phenotyping .

./ Measuring the NAT2 phenotyping status of UAE population samples .

./ Determining the NA T2 genotype frequencies of UAE population, and their

deviations from Hardy-Weinberg equilibrium .

./ Defining the correlation between NA T2 genotyping and phenotyping .

./ Discovering new mutations, identifying and classifying them if present. 60

Chapter 3: Methods

This study was approved by the Research Ethics Committee of the Faculty of Medicine and Health Sciences of UAE University (AI-Ain Medical District Human

Research Ethical Committee, AAMD/HREC No: 21 M059). No deviation from the protocol was implemented without the prior review and approval.

3.1 Research Design

3.1.1 Type of Study

Randomized, open labeled and mUlti-center study of the CYP1A2 and NA T2 polymorphism and their phenotyping and genotyping correlation in Emirati population, from the period of September 2012 till September 2016.

3.1.2 Setting

All the 7 emirates of UAE were involved; subjects were UAE National volunteers.

3.1.3 Including Criteria

Healthy subjects of more than 12 years of age.

3.1.4 Exclusion Criteria

Subjects who were smokers, not Emirati or aged less than 12 years were

excluded.

3.1.5 Place of Study

ates. The study was conducted in the United Arab Emir 61

3.1.6 Study Duration I Period of Study

The period of this study started from September 201 2 and ended in

September 2016 during the PhD study period.

3.1.7 Ethics and Ethical Approvals

Ethical approval for the research was taken by the Research Ethics

Committee of the Faculty of Medicine and Health Sciences of UAE University

(Appendix 1 and 2: AI-Ain Medical District Human Research Ethical Committee ,

AAMD/HREC No: 21 M059).

Communication letters were prepared and submitted for all the places that we were planning to visit for samples collection. Approval letters were collected from that places that had been visited (Appendix 3: UAEU Approval, Appendix 4: Abu

Dhabi Education council (ADEC) Approval, Appendix 5: Dubai Education zone

Approval and Appendix 6: Ras AI Khaimah Education Zone Approval). The volunteers signed a written consent form that their participation is voluntary

(Appendix 7: Consent Form).

3.1.8 Sampling

Individuals with United Arab Emirates nationality had been chosen who were above 12 years old, non-smokers and had been assigned randomly in a systemic way, from multicenters including all of the Emirates of UAE.

The assigned number of individuals who participated from each Emirate was defined based on UAE National Bureau of statistics of UAE (Emiratis) population

(Table 8). 62

Sample Size Calculation

The sample size calculation was carried out to ensure the precision of confidence interval to be 5%. Although, the frequencies of alleles were different from region to region we used sample size calculation assuming the maximum variability in data. By doing so, the sample size required for this prevalence study was around

500 subjects. The volunteers signed a written consent form that their participation was voluntary. To ensure that this number was achieved we recruited 581 subjects

(16% more subjects) to make up for incomplete data and withdrawals.

Sample Collection

Based on UAE population (Emiratis) distribution between the Emirates, the expected distribution of our samples was shown in following table (Table 8):

Table 8: The expected distribution of our samples based on the distribution of UAE population (Emiratis) between the Emirates

UAE emirates Population Samples expected to be collect Abu Dhabi (AD) 400000 = 42% 210 samples Dubai 171000 = 18% 90 samples Sharjah 152000 = 16% 80 samples Ras AIKhaimah 95000 = 10% 50 samples Fujairah 66500 = 7% 35 samples Ajman 47500 = 5% 25 samples Um AI Quwain 19000 = 2% 10 samples

Total 950000 = 100% 500 samples

3.2 Data Collection

Data collection form was filled by each volunteer who agreed to participate and signed the consent form only (Appendix 8: Data Collection Form). This was followed by samples collection and treated as anonymous since no names were disseminated. Finally, codes were assigned for each volunteer that had been used

in tracing the data in data collection and analysis. 63 3.2.1 Sample Collection

Five hundred and eighty one subjects were enrolled in this study. The subjects were asked Simply to consume 300ml of a caffeinated cola drink or a cup of strong tea or coffee an d then to provide a spot urine sample 2 hours later. No restrictions whatsoever regarding food intake or caffeine consumption are required of our subjects prior to their taking part in the study. However, volunteers were asked not to consume any of these beverages during the two hours period.

In addition, each volunteer had to provide buccal swab at the time of urine sample collection using Isohelix SK1 buccal swabs with Isohelix dri-capsules for sample collection (Figure 11). This buccal swab had a unique swab matrix that greatly improves DNA yields, it is an alternative to blood collection method, suitable for human, easy to handle and quick to use.

Puil Op n lh p. ck. fron, onil nd.

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PI" Q I.he """", b b"ck Into lh tub .1.>0 not tou h the hru�h wl rh YO ,-i,.hnq , ....

..... ;:)cC" your thumbn.:..' In the �rn,:, 11 q,oo\l !*C"t In lhC! h ..ndt .Ih n <.n•• p Ih '�"nd' In tWO by b ndln to on, �1t1 . ... . rh'" ",-\lV.-'" ht",.-u 1 ' ..')11 II 1.Co') rh tuh

• . , ..1':1 / Sc",' he tub '" urely wi h on of the cap"

Figure 11: Instruction of Isohelix SK1 buccal swabs use

3.2.2 Storage and Arrangement for Urine Samples and Buccal Swabs

Urine samples were placed on ice immediately after voiding and the pH of each sample was adjusted to <3.5 within 1 to 2 hours of collection to prevent loss of 64

5-acetylamino-6-formylamino-3-methyluracil (AFMU), a key metabolite of caffeine and other caffeine metabolites. After adjustment of pH, aliquots of urine were stored at -200 C until analysis. Sample analysis was done as described by Grant et al 1984 and Woolhouse et al 1997 as detailed in the following sections.

The collected buccal swab which consisted of Isohelix SK1 buccal swabs and Isohelix dri-capsule for each subject was placed immediately on ice after collection and then was stored at -200 C until analysis.

3.2.3 Determination of Phenotypes

3.2.3.1 The Extraction Protocol of Caffeine Metabolites from Urine

The urine samples were thawed to room temperature and vortexed thoroughly for 1 minute, ultrasonicated for 15 minutes and vortexed again for 5 seconds. Then, 0.2ml of urine and 120mg ammonium sulfate were added into a labeled clean 15ml centrifuge tube and vortexed for 30 seconds. This was followed by the addition of 6ml of chloroform: isopropanol (95:5) and vortexed for 1 minute.

The solution was centrifuged at 3000g for 5 minutes; the supernatant aqueous phase was carefully removed and dried in N2 flow at 42 °C. Finally, when the dryness of the solution was assured 0.5 ml of acetic acid (0.05%) was added to dissolve the contents.

3.2.3.2 Using HPLC Method

The mixture was filtered through 0.22u filter, 100 ul of the final product was

injected into the column of HPLC. Each sample took one hour to complete the cycle

on the HPLC.

High performance liquid chromatography (HPLC) was performed by

separating the metabolites using a reversed-phase 4.6x250 mm column (ultra- 6S sphere ODS-35uM, Hichrom, UK) and eluted with mobile phases A (0.045% acetic acid) and B (100% methanol) using a gradient profile starting 1 ml/min, 18% B at 0 minute and increasing to 50% B at 27 minute; increasing to 90% B at 32 minute;

90% B at 37 minute; reduced to 18% B at 42 minute till the end of 50 minute for re­ equilibration.

The standards included AFMU, 1 MU, 1 MX, 17MU, 17MX and 137MX.

Spectral conformation of caffeine metabolite peaks was performed routinely, using a programmable multiple wavelength detector (Waters 2998PDA, Waters Corp.,

Milford MA). Using this methodology, recovery of caffeine, paraxanthine, AFMU and

1-methylxanthine ranged from 91 to 103%.

Subsequently, the levels of caffeine metabolites including caffeine, AFMU,

17MX, 17MU, 1MU and 1MX were determined by HPLC.

Based on Butler et al (1992), caffeine 3-demethylation activity (CYP1A2) was assessed by determining the ratio (17MX + 17MU)/137MX. This molar ratio correlates well with the rate constant for hepatic 3-demethylation of caffeine based on a pharmacokinetic study (r= 0.73, P value = 0.007) compared to other urinary metabolism ratios 17MXl137MX, (AFMU+1 MX+1 MU)/1 7MU and

(AAMU+1 MX+1 MU)/17MU (Butler MA et al 1992). Since other studies had used this molar ratio, this measure was calculated and compared in this study, the frequency distribution of (17MX + 17MU)/1 37MX in our data indicated a trimodal distribution with specific cut points that represented slow, intermediate and rapid acetylators.

The ratio of (17MX+ 17MU)/137MX was calculated to determine the phenotype status of CYP1A2 activity in each subject as described by Tang B et al

1994 and Joshua EM et al 2008; the following table was used to identify the

CYP1A2 status (Table 9). 66 Table 9: Classification of CYP1 A2 enzyme activity based on the ratio of (17MX+ 17MU)/1 37MX

(17MX+ 17MU)/137MX ratio Enzyme activity status 0.7 2.5 Slow CYP1A2 enzyme activity 2.51-6.5 Intermediate CYP1A2 enzyme activity 6.51-above Rapid CYP1A2 enzyme activity

Caffeine 3-demethylation activity (NAT2) was assessed by determining the ratio of AFMU/1 MX. This molar ratio correlates well with the rate constant for hepatic

3-demethylation of caf feine based on a pharmacokinetic study (Butler MA et al

1992). This ratio typically falls into three phenotypes (slow, intermediate or rapid)

(Butler MA et al 1992). The ratio of AFMU/1 MX was calculated to determine the phenotype status of NAT2 activity in each subject as described by Grant OM et al

1984 and Woolhouse NM et al 1997. Table 10 was used to identify the acetylator status by calculation of AFMU/1 MX ratio.

Table 10: The acetylator status of NAT2 activity was classified based on the ratio of AFMU/1 MX

AFMu/1 MX ratio Acetylator status 0-0.5 Slow NAT2 acetylator 0.5-2 Intermediate NAT2 acetylator 2- above Rapid NAT2 acetylator

3.2.4 Determination of Genotypes

3.2.4.1 The Extraction Protocol of DNA from the Isohelix SK1 Buccal Swabs

DNA isolation protocol (ref: http://www. isohelix. com/documentation) was

carried out using Isohelix Buccal DNA Isolation Kit. Isohelix Buccal DNA Isolation

Kits were specifically formulated to produce high DNA yield and purity from buccal

swabs. The kits were fully optimised at cell projects LTD company (UK) for use on

buccal cell samples and offer reduced handling times, increased DNA yields and 67 many other Important technical benefits for their use in manual, 96-well or other high throughput formats.

Part 1: DNA Stabilization

Five hundred microliter Lysis buffer (LS solution) and 201-11 Proteinase K (PK solution) from kit were added to the tube containing the buccal swab and vortexed briefly. At this point the DNA was stabilized to proceed for DNA isolation or store the stabilized swab in a sealed tube at room temperature which can be stored for at least 3 � years.

Part 2: DNA Isolation

After DNA stabilization, the tube containing the swab, LS solution and PK solution was placed in a 60 °C water bath for 1 hour and vortex briefly. Then, the liquid in the tube (approximately 4001J1)was transferred into a 1.5ml centrifuge tube using a sterile pipette tip. Then, 4001J1 of Capture buffer (CT solution) was added to the tube and vortexed briefly and the tube was then placed in a micro-centrifuge

(with hinge positioned outwards so the liquid can be removed from the opposite side) and spun at maximum speed (13.4K rpm/12,000 x g) for 7 minutes to pellet the

DNA. Subsequently, all the supernatant were carefully removed with a pipette tip taking care not to disturb the DNA pellet. The tube was re-spun briefly and any remaining liquid removed as it was important to do this. Then, a small volume of re­ hydration buffer (TE solution) "-701J1" was added to the tube to dissolve the DNA.

The solution was left for at least 1 0 minutes at room temperature and vortexed briefly for the DNA to re-hydrate. Finally, the tube was re-spun for 15 minutes at maximum speed (13.4K rpm/1 2,000 x g) to remove un-dissolved debris. The supernatant was transferred to a sterile 1.5ml tube, being careful not to disturb the

pellet. 68 The DNA sample at this stage was ready for use in amplification. The storage of the DNA sample was at 4 °C for short term storage or at -20 °C for long term storage. The expected yield from a buccal swab was on average 1 to 10IJg

DNA (5 to 70ng/lJl) from an adult.

The purity and concentration of the DNA was ascertained by measuring the optical density of the DNA samples using Nano Drop Spectrophotometer.

At this stage and before starting PCR and during PCR, care was taken to avoid contamination as PCR assays require special laboratory practices to avoid false positive amplifications.

3.2.4.2 TaqMan Real Time PCR (Polymerase Chain Reaction)

TaqMan genotyping assays genotype single nucleotide polymorphisms

(SNPs) using the 5' nuclease assay for amplifying and detecting specific SNP alleles in purified genomic DNA samples. Each assay allows to genotype individuals for a specific SNP. Each TaqMan genotyping assay contains two primers for amplifying the sequence of interest and two TaqMan Minor Groove Binder (TaqMan

MGB) probes for detecting alleles. The presence of two probe pairs in each reaction allows genotyping of the two possible variant alleles at the SNP site in a DNA target sequence. The genotyping assay determines the presence or absence of a SNP based on the change in fluorescence of thedyes associated with the probes.

The TaqMan® MGB Probes consist of target-specific oligonucleotides with: first a reporterdye at the 5' end of each probe (VIC dye is linked to the 5' end of the

Allele 1 probe and FAM dye is linked to the 5' end of the Allele 2 probe) (Table 11).

Second, a MGB increased the melting temperature (Tm) without increasing probe

length (Afonina L et al 1997), thereby allowing the design of shorter probes; shorter

probes resulted in greater differences in Tm values between matched and 69 mismatched probes, resulting in accurate allelic discrimination. Third, a non­ fluorescent quencher (NFQ) was at the 3' end of the probe. Because the quencher did not fluoresce, Applied Biosystems Real Time PCR system could measure reporterdye contributions with great sensitivity.

The assays are supplied by Life Technologies "Applied Biosystems", USA.

The detailed procedure can be checked by TaqMan® SNP genotyping assays protocol booklet provided by Applied Biosystems.

Table 11: Fluorescence signal correlations

Fluorescence Increase Indication VIC dye Fluorescence only Homozygosity for allele 1 6FAM dye Fluorescence only Homozygosity for allele 2 Fluorescence signals for both dyes Heterozygosity for allele 1 and allele 2

Basics of the 5' Nuc lease Assay and during peR

Genomic DNA was introduced into a reaction mixture consisting of TaqMan

Genotyping Master Mix, forward and reverse primers and two TaqMan MGB Probes.

Each TaqMan MGB Probe annealed specifically to its complementary sequence, if present, between the forward and reverse primer sites. When the probe was intact, the proximity of the reporter dye to the quencher dye resulted in quenching of the reporter fluorescence primarily by Forster-type energy transfer (Lakowicz JR 1983).

The primers bounded to the template were extended by AmpliTaq Gold DNA

Polymerase which in turn cleaved the probes only that were hybridized to the target.

The cleavage separated the reporter dye from the quencher dye, which resulted in increased fluorescence signal by the reporter. Thus, the fluorescence signal generated by PCR amplification indicated which alleles were presented in the sample

(Figure 12). 70

sa Componen and D A empIa e e

F ,":a r: €

3.Signa Geoerc n

Figure 12: Complementary TaqMan probe fluorescenes after it anneals to the template and after cleavage by AmpliTaq Gold DNA Polymerase UP

CYP1A2 genotyping of all subjects was determined by TaqMan Real Time

PCR analysis to detect the five major observed CYP1 A2 mutations responsible for the phenotype (slow) of the corresponding allele described as small subset of G-

3860A, C-729T, 21 16G>A, 2499A>T and 5090C>T substitutions.

The G-3860A substitution was found in the CYP1 A2*1 C gene. It was located in the Sf-flanking region of human CYP1 A2 gene altering guanine (wild type) to adenine (mutated type) at position 2964 in the gene. The C-729T substitution was found in CYP1A2*1 K gene. it was located in the intron 1 region of the gene 71 changing cytosine (wild type) to thymine (mutated type) at position 873 in the gene as metioned earlier in table 3.

The 21 16G>A missense sUbstitution was found on CYP1A2* 3 gene cluster.

It was located in exon 4 causing Asp348Asn. The 2499A>T missense sUbstitution was found on the CYP1A2* 4 gene cluster. It was located in exon 5 leading to lIe386Phe. The 5090C>T missense substitution was found on the CYP1 A2*6 gene cluster. It was located in exon 7 leading to Arg431 Trp. The WT allele was formerly defined as the absence of G-3860A, C-729T, 21 1 6G>A, 2499A>T and 5090C>T nucleotide substitutions as metioned earlier in table 3.

The presence of these mutations "G-3860A, C-729T, 21 16G>A, 2499A>T and 5090C>T" were determined by specific designed assays. Each assay was able to genotype individuals for a specific SNP. Each TaqMan genotyping assay contained two primers for amplifying the sequence of interest and two TaqMan Minor

Groove Binder (TaqMan MGB) probes for detecting alleles. The presence of two probe pairs in each reaction defined the genotyping of the two possible variant alleles at the SNP site in a DNA target sequence. The genotyping assay determined the presence or absence of a SNP based on the change in fluorescence of the dyes associated with the probes.

In our assay, TaqMan genotyping assays were used to identify the important

SNPs that we investigated in CYP1A2 gene (rs2069514, rs1 2720461, rs56276455, rs72547516 and rs28399424). The assays with their IDs had been defined by the manufacturer company (Thermo Fisher Scientific) for each dbSNP with their specific primers that would detect a specific single nucleotide changes. Particularly, G-3860A,

C-729T, 21 16G>A, 2499A> T and 5090C>T that represented the major

SNPs/alterations responsible for the slow phenotype of the corresponding *1 C, *1 K,

*3, *4 and *6 haplotypes, respectively (Table 12). 72

Table 12: Assay 10 or design that has been used for each SNP

Forward and Reverse VIC dye- FAM dye- Primers Context Sequence fluorescence fluorescence Allele Assay dbSNP Haplotype 10 [VIC/FAM)" signal generated signal generated Nomenclature

G Wild type allele TGGCTCACCGCAACCTCC A At poSItion Mutant allele rs20695 GCCTCTC[G/A]GATTCAAG C 1585 3860 CYP1A2'1C 9191_30 14 CAATTGTCATGCCCCAG

C Wild type allele At position GGCTAGGTGTAGGGGTC T Mutant allele C -3063 rs12720 729 CTGAGTTC[CfT]GGGCTTT CYP1A2'1 K 4146_10 461 GCTACCCAGCTCTTGACT

G Wild type allele

At position GTACATGGGGGCCCCCA • A Mutant allele

C -3063 rs56276 21 16 ACCCTATA[G/AjACAGAGG CYP1A2'3 4247_20 455 AAGATCCAGAAGGAGCTG

A • Wild type allele At position ACACTCCTCCTTCTTGCC T Mutant allele rs72547 2499 C -3063 CTTCACC[AfT]TCCCCCAC CYP1A2'4 4246_10 516 AGGTGAGGCCTGCCGGT

GGACCCCTCTGAGTTCCG C • Wild type allele GCCTGAG[CfT]GGTTCCT At position T Mutant allele C -3063 rs28399 CACCGCCGATGGCACTG 5090 4244_20 424 C CYP1A2'6

Dote represent no signal has been generated

Storage and Stability

TaqMan Genotyping Master Mix is stable when stored at 2 to 8 °C.

Specific Guidelines

Particularly for TaqMan real time PCR, defining the method for adding DNA

reaction is important. Wet DNA delivery was selected where the aliquot genotyping

the final reaction mixed to an optical reaction plate and delivered genomic DNA to

mix. plate is also Nevertheless, proper selection of the instrument and reaction

signals generated by the essential in order to detect and record the fluorescent Biosystems Thermal Cycler cleavage of TaqMan probes. For that, Applied

™ Optical 96-well reaction plate; QuantStudio 7 Flex (278870068) with MicroAmp 73 reaction volume 20uL was used throughout the experiments. Figure 13 summarizes

the procedures for performing a genotyping experiment.

Finally. quantifying genomic DNA by adding 1 to 10 ng of DNA template per

reaction well to have a uniformed DNA concentration with all the samples and along

the experiment. Quantitate genomic DNA has been done using Nano Drop

Spectrophotometer machine for DNA quantification.

Prepare reaction mix

c:

.2.... C'O (J 1 � Prepare reaction plate 0. 'illllium WHi:

Perform PCR I or I Real-Time PCR mstrument Thermal cycler

Set up a new • plate read document 1 Perform post-PCR plate read 1 Real-Time PCR Instrument c o .;:: Anahp..ethe C'O plate read document C .- III .E .III- �- (J->­ III (OJ .- c: C

Preparing the Reaction Mix Guidelines

For optimal PCR performance: first, all TaqMan reagents were kept protected from light until they were ready to use as excessive exposure to light may affect the fluorescent probes. Second, freeze-thaw cycles were minimized. Third, prior to use: the TaqMan Genotyping Master Mix was mixed thoroughly by swirling the bottle, thawing any frozen TaqMan reagents by placing them on ice and when thawed, the samples were re-suspended by vortexing then centrifuging the tubes briefly. Finally, the PCR reaction mix was prepared for each assay before transferring it to the optical reaction plate for thermal cycling and fluorescence analysis.

The number of reactions were calculated to be performed for each assay with one extra reaction for each ten to provide excess volume for the loss that occur during reagent transfers. Then, the volume was calculated of each reaction mix component needed for each assay "20ul reaction for each sample using 96-well standard plate".

The reaction plate was prepared using wet DNA delivery method by pi petting the reaction mix into each well of a reaction plate, inspecting each well for volume uniformity, noting which wells do not contain the proper volume, covering the plate with MicroAmp Optical Adhesive Film, centrifuging the plate briefly to spin down the contents and eliminating any air bubbles from the solutions, diluting 1 to 10 ng of each purified genomic DNA sample into DNAase-free water, pi petting samples into the plate, covering the plate with MicroAmp Optical Adhesive Film or MicroAmp

Optica l Caps and centrifuging the plate briefly to spin down the contents and eliminate air bubbles from the solutions.

Later, amplification of genomic DNA was performed by the Sequence

Detection System (SDS) using the thermal cycling condition that specified and 75 optimized to be used with TaqMan genotyping assay only (Figure 14) where 1 cycle denaturation at 60°C initially for 30 sec, then held at 95°C for 10min, then 40 cycle

(denature at 95°C for 15 sec and anneal/extend at 60°C for 1 min), then 1 cycle at

60°C for 30. Standard mode thermal cycling setting was selected in the pla te document (Figure 15), the reaction volume was settled accordingly and the reaction plate was loaded the thermal cycler to start running.

-- -

\ .

...

Figure 14: The thermal cycling condition specified for our TaqMan genotyping assays for CYP1A2

If, 1 ..,...... ' .... " � •• " ...... ' 1' .�, �", ."".,,-, II I 1'"" I� , .. ..' ..r '•. II.V ....·...... a-

_'... l """ _...... _'

_ ...... ' ...... vaf '

ting Figure 15: The Standard mode thermal cycling set 76 Allelic Discrimination Plate Read and Analysis

After peR amplification, an endpoint plate read was performed on a Real

Time peR instrument ( pplied Bio y terns Thermal Cycler Quant tudio 7 Flex

mod I no. 278870068) using the fluorescence measurements (Rn) made during the

plate read and the SOS software plots Rn values based on the fluorescence signals

from each well.

The plotted fluorescence signals indicated which alleles were in each sample

"The SOS software plots showed the results of the allelic discrimination run on a

scatter plot of Allele 1 versus Allele 2. Each well of the 96-well plate was

represented as an individual point on the plot" (Figure 16). The alleles were

determined in each sample as following: first, the allelic discrimination plate read

documents were created and set up (Figure 17). Second, post-peR allelic

discrimination plate read was performed on a real time peR instrument (Figure 18).

Third, the plate read documents were analyzed. Finally, an automatic or manual

allele calls and allele types verified were made.

23

t·• Homozygous or Allele 2 16

cr:- � 1.3 Heterozygous \b. N

• � 0.8 ... Homozygous forAlle.e • o Templa:e Control 0.3

1 . 1 3 5 01 03 05 07 09 1

AI Ie C) R

Figure 16: Variation in clustering due to the genotype of the target allele 77

Figure 17: The allelic discrimination plate read document

Figure 18: RT-peR plate layout during sample running in the system 78 The CYP1A2*1 A haplotype represented the wild type allele whereas CYP1A2*1C, CY P1A2*1K, CYP1A2*3, CYP1A2*4 and CYP1A2*6 genotypes repr esented the mutant alleles. Individuals who were homozygote for CYP1A2 polymorphisms (*1 C, *1K, *3, *4 and *6) were classified as slow phenotype.

Individuals who were heterozygote for CYP1A2 polymorphisms had an intermediate phenotype. Individuals who lacked CYP1A2 polymorphisms had a extensive or normal phenotype.

3.2.4.3 Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR - RFLP)

3.2.4.3.1 Polymerase Chain Reaction (PCR)

Amplification of genomic DNA was performed by the polymerase chain reaction (PCR) using the pair of forward and reverse oligonucleotide primers of

NA T2, master mix that is specified for NA T2 amplification and to be amplified at certainco nditions as described by Hickman & Sim (1991).

Reagents for peR

Tenfold PCR buffer concentrate, including magnesium chloride (15mM), was supplied by the manufacturer Bioline of the DNA Taq polymerase, the Q solution,

Deoxynucleoside triphosphates (dNTPs), restriction enzymes: Mspl, Ddel, Fokl, kpnl, Taql and BamHI, restriction enzyme buffer 10-fold concentrate supplied by the manufacturer of the restriction enzymes, sterile water for the amplification and themostable DNA Taq polymerase supplied by the manufacturer.

The reagents were stored at -20 °C and the work was done on ice through all

cycler. the procedures, the PCR tubes were kept on ice until placed in the thermal degree of The Q solution facilitates amplification of templates that have a high behavior of DNA. secondary structure or that are GC-rich by modifying the melting 79

The PCR buffer provides a final concentration of 1 .5mM MgCI2 in the final reaction mix which provided satisfactory results.

Amplification of 442bp and 559bp Segments of NA T2 Gene Sequence

Genomic DNA was obtained from the buccal swab samples of subjects by specific procedure for extraction. DNA amplification was performed by thermocycler

GeneAmp® PCR system 9700 version 3.12 using 0.1-0.5 IJM of oligonucleotide primers (forward primers; 5/-GTC ACA CGA GGA M T CM ATG C-3' and 5/-ACA

CM GGG TTT A TT TTG TTC C-3/) 18.6nmol/100ul and (reverse primers; 5/-ACC

CAG CAT CGA CM TGT M T TCC TGC CCT CA-3' and 5/-MT T AC ATT GTC

GAT GCT GGG T-3/) 11.03nmol/1 00ul described by Hickman and Sim (1991) in a

20 IJI mixture of 2ul of 10X solution (PCR buffer contain 15mM MgCI2, 4ul of Q solution (5X), 0.2ul of dNTP solution 200uM, 11 ul H20 (sterile water for amplification), 0.2ul of Taq DNA polymerase 2.5 units/reaction, 0.8ul of each primer and 1 ug of the sample DNA. Reagents and mixture were kept on ice during preparation. Negative control (DNA not added) was used for quality control.

The 10X buffer, dNTP mix, primer solutions, Q-solution, sterile water for amplification and Taq DNA polymerase were thawed on ice and the master mix was kept on ice after complete thawing and mixed thoroughly before use to avoid localized differences in salt concentration. The reaction mix was prepared accordingly and mixed gently but thoroughly by pipetting up and down a few times.

Then, the template DNA ($ 1 ug/reaction) was added to the individual PCR tubes containing the reaction mix. Finally, amplification was done using GeneAmp® PCR system 9700 version 3. 12. Amplification conditions were denaturation 1 cycle at

94°C for 5 min, then 35 cycles (94°C 30 sec, 56°C 1 min, 72°C 2 min), then 1 cycle

72°C 7 min, then cooling at 4°C (Figure 19). 80

Figure 19: The thermocycler program for the NA T2-PCR

Reagents for Agarose Gel Electrophoresis

Agarose, bromophenol blue, ethidium bromide "store in dark place", Boric acid (H3B03), ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) and tris-hydroxymethylaminomethane (TRIS). The last three were used to prepare the

TBE buffer concentrate (Table 13). The agarose gel was prepared by dissolving agarose in TBE solu tion in different concentrations based on the needs. One percent gel conc entration was prepared to run the control PCR before digestion.

One percent, 1.5% and 2.5% gel concentration were prepared to run PCR after digestion with certain enzymes. Then, the gel was placed in electrophoresis machine with addition of Ethidium bromide (0.5ug/ml) 5uL.

The 100bp DNA ladder (supplied by Promega Corporation, USA) was composed of bands where the fragments of DNA samples would be compared to. It consisted of eleven double-stranded DNA fragments with sizes of 100, 200, 300,

400, 500, 600, 700, 800, 900, 1000 and 1500bp. The int ensity of these fragments appeared to be equal on the gel with an exception of the 500bp band which showed triple the intensity of other fragments. Accordingly, the DNA ladder and DNA samples were added into specific wells to be run in the gel for certain time (60-90 minutes) under 80-100 V cm-1 . 81 Table 13: Preparation of TBE solution

Reagent Volume 2L 1L 500mL Boric acid 110g 55g 27.5g TRIS 216g 108g 54g E?TA 14. 8g 7.4g 3.7g Dissolve TRIS and EDTA first then add Boric acid. Make up to volume with distilled water

3.2.4.3.2 Restriction Fragment Length Polymorphism (RFLP)

In molecular biology, restriction fragment length polymorphism (RFLP) is a

technique that exploits variations in homologous DNA sequences. It refers to

a difference between samples of homologous DNA molecules from different

locations of restriction enzyme sites. In RFLP analysis, the DNA sample was broken

into pieces (digested) by restriction enzymes and the resulting restriction

fragments were separated according to their lengths by gel electrophoresis. NA T2

genotyping of all subjects was carried out by PCR-RFLP analysis as described by

Vatsis KP et al (1995), Woolhouse NM et al (1997) and Chida M et al (1999).

NA T2 genotype of all subjects was determined by PCR-RFLP analysis to

detect the four commonly observed NA T2 mutation associated with slow acetylation

usually described as minor allele of 191G>A (rs1801279), 341 T>C (rs1801 280),

590G>A (rs1799930) and 857G>A (rs1799931). The 341 T>C (rs1801280)

substitution is found in NA T2*5 gene cluster. The 590G>A missense substitution is

found in NA T2*6 gene cluster. The 857G>A missense substitution is found on

NA T2*7 gene cluster. The 191 G>A missense substitution is found on the NA T2*14

gene cluster (Vats is K P et al 1995). The WT allele was formerly defined as the

absence of 341T>C, 590G>A, 857G>A and 191G>A nucleotide substitutions.

The presence of these mutations "341 T>C, 590G>A, 857G>A and 191 G>A"

were determined by restriction endonuclease digestion using Ddel, Taql, BamHI and

Mspl, respectively. Then, the presence of 282C>T, 481C>T and 803A>G were 82 determined by rest riction endonuclease digestion using Fokl, Kpnl and Ddel respectively to identify the gene cluster or subtype. After amplification of DNA, digestion of the PCR product (1 ug - quantitate genomic DNA has been done using

Nano Drop Spectrophotometer machine for DNA quantification.) was carried out in a total volume of SOul using the appropriate digestion buffer (Table 14). The PCR product obtained using aforementioned primers was digested in separate experiments for 6hrs at 37° with Kpnl, Ddel, Fokl, BamHI and Mspl and for 8hrs at

6S0 with Taqi. Digested DNA (10ul) then was separated by electrophoresis on 1% for Mspl restriction enzyme, 1.S% for Ddel, Fokl, Kpnl and BamHI restriction enzyme and 2.S% for Taql restriction enzyme agarose gel under 80-1 00 V cm-1

(Table 14). DNA bands were visualized with ethidium bromide and UV transillumination.

Table 14: The restriction cleavage for the amplificates of the PCR product

Volumes for one sample Kpnl Taql BamHI Mspl Ddel Fokl PCR product 1ug 1 ug 1 ug 1ug 1ug 1ug Master mix Restriction enzyme 1 ul 1 ul 1 ul 1 ul 1 ul 1 ul 10X NE Buffer Sui Sui Sui Sui Sui Sui Total Rxn Volume SOul SOul SOul SOul SOul SOul Incubation 37° C 6So C 37° C 37° C 37° C 37° C Temperature Incubation Time 6hrs 8 hrs. 6hrs 6 hrs. 6 hrs. 6 hrs. Can also be used overnight with no star activity Agarose gel to run the 1.S% 2.S% 1.S% 1% 1.S% 1.S% samples

Primers

The pairs of forward and reverse oligonucleotide primers were (forward primers; S'-GTC ACA CGA GGA M T CM A TG C-3' and S'-ACA CM GGG TTT

ATT TTG TTC C-3') and (reverse primers; S'-ACC CAG CAT CGA CM TGT MT

TCC TGC CCT CA-3' and S'-M T TAC ATT GTC GAT GCT GGG T -3') as described 83 by Hickman & Sim (1991) respectively were used to amplifying 442bp and 559bp segment of NA T2 gene sequence. The 442bp fragment was used for 191 G>A, 282C> T, 341 T>C and 481 C>T mutations detection, whereas the 559bp fragment was used for 590G>A, 803A>G and 857G>A mutations detection.

The primers were dissolved in sterile water so that concentrations of

100pmol/ul were available. They were stored at -200 C. The primers were stable for approximately 1 to 2 years under these conditions.

DNA Sequencing

DNA sequencing was done uSing Applied Biosystems 3130xl Genetic

Analyzer machine for certain alleles that were not analyzed based on the designed evaluation sheet described below (Table 16). DNA sequencing was done for certain samples randomly as a confirmation of the accuracy of our technique. The gene accession number is NG_012246.1 and the transcript accession number is

NM 000015.2. CDS 13760 -14632.

Nomenclature

Using RFLP analysis, the presence/absence of polymorphism(s) in each

DNA sample was ide ntified. Accordingly, the genotype was determined as per the last update of NAT nomenclature scheme published by The Arylamine N- acetyltransferase Gene Nomenclature Committee according to consensus guidelines (http://nat.mbg.duth.gr/Human%20NAT2%20alleles 2013.htm).

and Restriction Cleavage Allelic Linkage Analysis - Restriction Map of NA T2 Site

trans­ On completion of electrophoresis, the wet gel was placed on the UV

excited to cause illuminator and the ethidium bromide intercalated in the DNA was photographed with the fluorescence emission at 312 nm. The band pattern was 84 camera of the evaluation device and printed out. The results for the investigated sample were recorded by marking the pertinent field in an evaluation sheet (Table 16) for each allele variation detected in the DNA fragments (Table 15). The allele combination in the samp les can be determined by superimposing the evaluation scheme (Table 16) of the same scale. The evaluation is complete, when the variations specified on the scheme can be assigned to ali points (reference sequence = wild type or sequence variation = mutant). If only one row on the eva luation scheme showed a match with the entries in the evaluation row, the

person was a homozygous allele carrier. If two rows of the evaluation sheet

matched with the evaluation scheme rows, the person was a heterozygous allele carrier. If it was impossible to determine which of the alleles concerned was really

present, DNA sequencing was done to identify these alleles.

DNA fragment lengths from the restriction cleavage has been illustrated by the following table (Table 15 and Figure 20) where the reference sequence (RS) = wild type, sequence variation (SV) = mutant and the control peR = 442bp and

559bp.

Table 15: The restriction cleavage site and its DNA fragment lengths from the restriction cleavage

Restriction cleavage Mutation site Sequence - fragment lengths (bp) Mspl 191 RS - 181, 168, 93 SV = 274, 168 Fokl 282 RS = 337, 105 SV = 442 Odel 34 1 RS = 221, 163, 58 SV = 189, 163, 58, 32 Kpnl 481 RS = 424, 135 SV = 559 Taql 590 RS = 226, 170, 142, 21 SV = 396, 142, 21 Odel 803 RS = 345, 124, 90 SV = 345, 97, 90, 27 BamHI 857 RS = 515, 44 SV - 559

First PCR = 442 Second PCR 559 85

Exon 1 Intron1 Exon 2 -12 15bp

NAT2

100bp 5!UTR \ 3' UTR 191 G>A 282C>T 341 T>C 481C>T 690G>A B03A>G 857G>A

Figure 20: NA T2 gene structure with the mutation sites

The *4 allele represents the wild type allele whereas *5, *6, *7 and *14 haplotypes represent the mutant alleles. Individuals who were homozygous for

NA T2 polymorph isms (NA T2 *5, NA T2*6, NA T2 *7 and NA T2 *14) were classified as slow acetylator phenotype. Individuals who were heterozygous for NA T2 polymorphisms had an intermediate acetylator phenotype. Individuals who lacked

NA T2 polymorphisms had a rapid acetylator phenotype.

Allelic Linkage Analysis - Restriction Enzymes

Evaluation sheet ( = fragment length combination found) and restriction enzymes with mutation sites and fragment length for NA T2 have been documented in the following table (Table 16) where the control peR = 442 bp and 559 bp to show how we assigned NA T2 alleles. Nevertheless, examples of fragment lengths found for 15 samples have been clearly illustrated as guidance. 86 Table 16: Evaluation sheet ( or · = fragment length combination found) of the restric tion enzymes with mutation sites and fragment length for NA T2

First peR = 442 Second peR = 559 Mspl(191) Fokl (282) Ddel (341) Kpnl (481) Taql (590) Ddel (803) 9amHI (857) WT Mu WT Mu WT Mu WT Mu WT Mu WT Mu WT Mu First peR = 442 bp Fragment 181 274 337 442 221 189 424 559 226 396 345 345 515 559 Second peR 559 lengths (bp) bp after 168 168 105 163 163 135 170 142 124 97 44 restnctJon 93 58 58 142 21 90 90

32 21 27

Haplotype Examples of fragment lengths

'4 Wild type allele

A 'S Mutant allele

'58 Mutant allele

'SC Mutant allele

'50 Mutant allele

'M Mutant allele

'68 Mutant allele

' 78 Mutant allele

'14A Mutant allele

'148 Mutant allele

Sample code Examples of fragment lengths found for 15 samples Assigned NA T2 allele combinations '41*4 (wUwt) homozygote

2 v '41'5A (wUmu) heterozygote

3 v '41'6A, '131'68 (wUmu) heterozygote 4 ,I '4/'78 (wUmu) heterozygote

5 '4i'14A (wUmu) heterozygote

6 'SAI'58 (mulmu) homozygote '581"6A, '5A1'6C 7 (mulmu) heterozygote '59/'78 (mu/mu) 8 v heterozygote '581'14A (mulmu) 9 heterozygote '6A1'6A (mu/mu) 10 homozygote '6A1'79 (mu/mu) 11 v heterozygote '6A1'148 (mu/mu) 12 heterozygote .J '781"78 (mu/mu) 13 homozygote

v '781'148 (mulmu 14 heterozygote '1481* 148 (mulmu) 15 homozygote 87 3.3 Data Analysis

The CYP1A2 haplotype construction and analysis was performed using

QuantStudioTM 6 and 7 Flex Real Time PCR System Software v1 .0 thorough

Thermo Fisher Cloud Scientific Analysis, instrument type: QuantStudio 7 Real-Time

PCR System. Slow and rapid CYP1A2 genotypes were reported according to the

Human CYP-Allele Nomenclature Committee.

The NA T2 haplotype construction and analysis was performed with the

PHASE v2.1.1 program (Stephens M et al 2001, Agundez JA et al 2008 and Selinski

S et al 201 3a) using the default model for recombination rate variation (Li N and

Stephens M 2003). Seven independent runs with 1000 iterations, 500 burn-in iterations, and a thinning interval of 1. The run that showed the maximum consistency of results across all runs was selected to be the best run, as described by Agundez et al. 2008. Slow and rapid NAT2 genotypes were reported according to the consensus NAT2 gene nomenclature (Hein OW et al 2008).

Data entry analysis SPSS (Statistical Package for the Social Sciences) version 19 was used for data entry and analysis. Kruskal-Wallis test, Spearman's rho and other statistical tests were used for analysis like Paired sample T test,

Pearson Chi-Sequare, Likelihood Ratio, Regression, Phi, Cramer's V, Odds Ratio and Relative Risk tests. A p value of 0.05 was considered statistically significant.

The deviation from Hardy-Weinberg Equilibrium was also calculated for each genotype to determine whether observed genotype frequencies are consistent with

Hardy-Weinberg equilibrium using Chi-Sequare test. 88

Chapter 4: Results

4.1 Population Data

The study was carried out on 556 non-smoker Emirati males ("95% - mean

age 18 years") and 25 females ("5% - mean age 25 years"). Five of the recruited

(-1 %; 3 males and 2 females) had been assigned to standardize the procedures

and protocols before proceeding with the study samples and 576 subjects (99%;

553 males and 23 females) were assigned as study samples.

The age ranged from 16 - 51 years old; the vast majority of the recruited

subjects were between 16 and 19 years of age. (Figure 21).

A ge Distribution 200 180 >- 160 u C 140 Q.> :::;, 120 � 100 u- 80 60 40 20 0 16 17 8 19 20 21-30 >�o Age

Figure 21: The age distribution of the recruited subjects

All of our subjects were from UAE population who were originally from this

region. Out of all subjects, 269 (46.3% of the total recruits) were from Abu Dhabi

Emirate (96.6% of them were males and 3.4% females), 113 (19.4%) were from

Dubai Emirate (95.5% males and 4.5% females) and 199 (34.3%) were from

Northern Emirates (94.4% males and 5.6% fe males). and the areas The distribution of subjects who were enrolled in this study

where samples were collected represented almost the same distribution of UAE

population (Emiratis) between the Emirates. There is no significant difference in 89 sample distribution between the Emirates compared to the data provided by UAE

National Bureau of statistics of UAE (Emiratis) population (P = 0.262; using 95% confidence interval of differences) using Paired Samples T test.

4.2 Descri ptive Analysis of the Recruited Sample

Out of all sample subjects 290 (50.3%) were children of consanguineous marriages reflecting the high of consanguinity among Emiratis. Out of those 290 subjects, 185 subjects (63.7% "181 males and 4 females") had third degree related parents and 105 (36.3% "101 males and 4 females") had the same tribe or family name. On the other hand, 286 subjects (49.7%"271 male and 15 females) were children of non-relative parents.

Out of all sample subjects 520 (90% "499 males and 21 females") were healthy whereas 56 (10% "54 males and 2 females") had medical illness. Out of those with history of illness 3 subjects (0.6%) had allergic conditions, 4 subjects

(0.8%) had cardiovascular disease, 23 subjects (4%) were with asthma, 2 subjects

(0.4%) had epilepsy, one subject (0.2%) had hypercholesterolemia, one case (0.2%) had vertebral disc disease, 4 subjects (0.8%) had diabetes mellitus, 16 cases (2.9%) had Sickle-cell disorder, one (0.2%) was with migraine and (0.2%) had thyroid disease.

Out of all sample subjects, 555 (96.3%; 532 males and 23 females) reported no sensitivity to medications whereas 21 (3.7%; "; 21 males) subjects had reported sensitivity to medications. Out of them 2 cases (0.3%) had antibiotics allergy (not specified by the participants), 6 subjects (1 %) had sensitivity to G6PD deficiency related drugs, 2 cases (0.3%) were sensitive to ibuprofen and 11 cases (1.9%) had allergies of unknown cause. 90 4.3 HPLC Analysis

High performance liquid chromatography (HPLC) was performed by

separating the metabolites included AFMU, 1 MU, 1 MX, 17MU, 17MX and 137MX,

and their levels were determined based on the area under the curve that has been

specified through specific retention time for each metabolites peak (AFMU = 3.182,

1MU = 4.868, 1MX = 5.812, 17MU = 8.279, 17MX = 8.588 and 137MX = 12.440)

measured by specific standards (Figure 22).

u'v'

o U5 => x x �v � � ! � ::� 0,,,, �- a>� "'" � 012 =0 �""

� - 010 ..., ;: oos

000

I 00-.<

OO:? I '" 000 '" � - .1,.. � \

000 2.00 .a 00 600 800 000 '2 00 ,-1.00 '6 00 1800 20Q 1l-f'tuifiG. 1 Figure 22: Representative chromatogram curves that are specific for caffe ine metabolites with their retention time

The standards included AFMU, 1 MU, 1 MX, 17MU, 17MX and 137MX.

Spectral conformation of caffeine metabolite peaks was performed routinely, using a

programmable multiple wavelength detector (Waters 2998PDA, Waters Corp.,

Milford MA). Using this methodology, recovery of caffeine, paraxanthine, AFMU and

1-methylxanthine ranged from 91 to 103% (Figure 23, 24, 25, 26, 27 and 28). 91

,. %..

... r .% _5:

Figure 23: Standard Spectral conformation of 137MX, 17MX, 17MU, 1 MU, 1 MX and AFMU. The amount on X axis is in ug/ml 92 4.4 Statisti cal Analysis

4.4.1 CYP1A2

4.4.1 .1 CYP1A2 Genotype

Genetically, we were able to genotype all cases for the selected polymorphism using TaqMan Real Time peR analysis. We found out of 576 subjects 8 (1 .4%) subjects were homozygote for mutant alleles, 93 (16.1 %) were heterozygote and 475 (82.5%) homozygous for the wild type allele genotyping

(Figure 24).

HO"'OZV8ote Heteroz.vaote Hon'lOZV8ote ",ut.nat all .. 1... \Nlld 'TVpe

Figure 24: The genotype frequencies of the studied sample.-0.014 of our subjects were homozygote for mutant alleles, 0.161 were heterozygote and 0.825 were homozygous for the wild type genotyping

The overall mutant alleles' frequency was 0.095 with allele CYP1A2*1C frequency = 0.0636, allele CYP1A2*1 K = 0.0287, allele CYP1A2*3= 0.0027, allele

CYP1A2*4= 0 and allele CYP1A2*6 = 0 while the wild type allele frequency was found to be 0.905 (Figure 25). 93 "U I. , n ., "

Figure 25: Allele frequencies- allele CYP1A2*1C frequency = 0.0636, allele CYP1A2*1K = 0.0287, allele CYP1A2*3= 0.0027, allele CYP1A2*4= 0 and allele CYP1A2* 6= O. The wild type allele frequency was found to be 0.905

The follo wing figure (26) shows an example of allelic discrimination plot that

demonstrates a variation in clustering due to the genotyping of the targeted allele ™ (CYP1A2*1C) using QuantStudio 6 and 7 Flex Real Time peR System Software

v1 .0.

� - , . c D::

ii, + �" tl�Ir-t ' ,, � " N il CI> Gi

II

lioOlO OU (,. t t \ 11d I ... , u , \J"l�' • fuill .t . .- .... .

" , " . Oft II , , , , . , " I , ,u Allele 1 (VIC) Rn "G" Figure 26: An example of allelic discrimination plot that demonstrates a variation in clustering due to the genotyping of the targeted allele (CYP1 A2*1 C)

We are able to identify six different CYP1A2 diplotypes. Out of the 576

subjects, 568 (98.6%) subjects were homozygote and heterozygote for the wild type

allele". Of whom, 475 (82.5%) subjects were homozygote for the wild type allele, 59 94

(10.2%) subjects were heterozygote for CYP1A2*1A1*1C genotype, 33 (5.8%) were heterozygote for CYP1A2*1A1*1K genotype and 1 (0.2%) was heterozygote for

CYP1A2*1A1*3 genotype. Whereas, 8 (1 .4%) subjects were homozygote for the mutant alleles. Of whom, 7 (1.2%) subjects were homozygote for allele CYP1A2*1C and 1 (0.2%) subjects was homozygote for allele CYP1A2*3 (Table 17).

Table 17: The CYP1A2 genotype frequencies and distribution

CYP1 A2 genotype n Frequency CYP1A2*1A1"1A 475 0.825 CYP1A2*1 Al"1 C 59 0. 102 CYP1A2*1A1"1K 33 0.058 CYP1A2*1 Al"3 1 0.002 CYP1A2*1CI*1C 7 0.012 CYP1A2*31*3 1 0.002

The characteristic features and frequencies of the various CYP1 A2 haplotype identified among Emiratis, determined from TaqMan Real Time peR analysis of genomic DNA is shown in table 18. The most common haplotypes by far among our population sample belonged to the CYP1A2* 1A (rs2069514) gene and occurred with a frequency of 0.905. Three different CYP1A2 alleles associated with slow activity were found. The frequencies of these slow alleles CYP1A2*1C, CYP1A2*1K and

CYP1A2*3 were less common than wild type allele, the allele belonged to the

CYP1A2*1C (rs12720461) gene occurring with a frequency of 0.0636. Whereas, the frequency of CYP1A2*1 K (rs56276455) and CYP1A2*3 (rs72547516) genes were negligible 0.0287 and 0.0027, respectively. The frequency of CYP1A2*4

(rs28399424) and CYP1A2*6 (rs2069514) alleles were absent in this population

(Table 18).

While examining the deviation from Hardy-Weinberg equilibrium, it was also found that the observed genotype frequencies were different from the expected one. nonetheless, the data for CYP1 A2*1 K were in accordance with the Hardy-Weinberg equilibrium since P>0.05. For CYP1A2*1C, the data did not obey the equilibrium 95 since P = 0.001. CYP1A2*3, CYP1A2*4 and CYP1A2*6 were too rare for the equilibrium to be determined (Table 18).

Table 18: The characteristic features and frequencies of the various CYP1 A2 haplotypes identified among Emiratis Haplotype dbSNP Enzyme n Percentage Hardy-Weinberg Equilibrium activity *1A rs2069514 onnal 1043 90.5% *1C rs1 2720461 Decreased 73 6.36% Var. allele freq .= 0.07, P=0.001 *1 K 33 2.87% Var. allele freq.= 0.03, P=0.44 rs56276455 Decreased

*3 rs72547516 Decreased 3 0.27% Can't be counted* *4 rs28399424 Decreased 0 0% Can't be counted* *6 rs2069514 Decreased 0 0% Can't be counted* Var. allele freq. - Variant allele frequency *Not accurate if <5 individuals in any genotype group.

Based on epidemiological studies, it has been clearly observed that there is

a significant ethnic variability in the distribution of common and rare CYP1A2 SNPs

and haplotypes (Zhou SF et al 2009b). The available data suggested that the

frequency of CYP1A2*1C allele was 0.009 in British, 0.008 in Swedish, 0.23 in

Japanese, 0.26 in Korean, 0.22 in Chinese, 0.07 in African American, 0.07 in

Tunisian, 0.04 in Turkish and 0.07 in Egyptian populations (Nakajima M et al

1999, Chida M et al 1999, Hamdy SI et al 2003, Tiwari AK et al 2005, Bilgen T et al

2008). In the contrary, based on our study; the Emirati population was one of the

lowest frequencies with 0.06 when compared to most of the mentioned populations.

Taking in consideration, the number of the subjects used in our study was the

hig hest in contrast to other studies (Figure 27A) .

Furthermore, the frequency of CYP1A2*1K allele was 0.003 in Swedish,

0.005 in Spanish, 0.04 in Saudi Arabian and 0.03 in Ethiopian populations, whereas

it was absent in Korean and Japanese populations (Aklillu E et al 2003, Ghotbi R et

al 2007). Likewise, the genotype frequency of CYP1A2* 1 K haplotype in Emiratis 96 population was slightly lower than Saudi Arabian and Ethiopian population with

frequency of 0.02 (Figure 278).

Lastly, the genotype frequencies of CYP1A2*3, CYP1A2*4 and CYP1A2*6 alleles have been identified in the French population with frequency of

(0.005-0.01) (Chevalier et al 2001, 0 Allorge 0 et al 2003, Zhou H et al 2004). On the contrary, the frequencies of these haplotypes were lower in the Emirati

population specifically the haplotype CYP1A2*3 frequency, that was found to be

0.002. Similarly, the frequencies of CYP1A2*4 and CYP1A2*6 haplotypes were absent in Emiratis population (Figure 27C).

(27A) 02'0.

o 1 t;

01

o.os

(27B) O.OJ5

004

0.035

0.03

0.025

002

0.015

--- 0.01

0.005

a [mlf.Jlls SWcdC5 Spaniards Koreans 5.Judl [thlOPI.JflS Japanese Arablan� 97

(27 ) 00]2

0.01

o OOS

0006 Ii r'l!ndl

0.004

0.001

CYP JAl"'l C'(PI AJ" Ii

Figure 27: The frequency of CYP1A2 alleles among Emiratis in comparison with other populations, figure (A) shows the CYP1A2 *1 C haplotype frequencies, figure (B) shows the CYP1A2*1 K haplotype frequencies, figure (C) shows the CYP1A2*3, CYP1A2*4 and CYP1A2*6 haplotypes frequencies, n = sample size, Emiratis (n = 576), Japanese (n = 250), Koreans (n = 150), Chinese (n = 168), African Americans

(n = 112) , Tunisians (n = 98), Turkish (n = 110), Egyptians (n = 212) , Swedish (n = 194), French (n = 100), British (n = 114), Spaniards (n = 117), Saudi Arabians (n = 136), Ethiopians (n = 173)

4.4.1.2 Caffeine Metabolites Ratio and CYP1 A2 Phenotype Status

Since other studies had used "(17MX + 17MU)/1 37MX" molar ratio, this measure was calculated and compared in this study (Butler MA et al 1992 and, Lang

NP and Kadlubar FF 1991). The frequency distribution of (17MX + 17MU)/1 37MX in our data indicated a trimodal distribution with specific cut points 2.5 and 6.5 that represented slow, intermediate and rapid acetylators (Joshua EM et al 2008).

Out of 576 subjects, 560 showed positive results whereas the remaining 16 did not complete the study. From those 560 subjects, 8 (1.4%) were slow CYP1A2 enzyme activity "ratio < 2.5", 91 (16.3%) were intermediate activity "ratio between

2.5 -2.6" and 461 (82.3%) were rapid activity for this enzyme "ratio > 6.5" (Table 19). 98

Table 19: The classification and the results of the CYP1A2 enzyme activity based on the ratio of (17MX+ 17MU)/1 37MX (17MX + phenotype status No. of Percentage 17MU)/137MX ratio subjects

0.7 2.5 Slow CYP1A2 activity 8 1.4%

2.51-6.5 Intermediate CYP1A2 activity 91 16.3%

> 6.51 Rapid CYP1A2 activity 461 82.3%

The ratio of (17MX + 17MU)/1 37MX ratio was calculated to determine the phenotype status of CYP1A2 activity in each subject. The frequency distribution histogram of urinary (17MX + 17MU)/1 37MX molar concentration ratio for 560 UAE nationals shows non-normally distributed result "Median = 5.05, Skewness = 1.215 with Std error = 0.1 03, Kutosis = 1.239 with Std error = 0.206" (Figure 28).

80 70 ." bO :Ei ('l iii 40 0 30 ci ;?O z 10 I . • - - 0 I � w. � \.(\ � �

Figure 28: The distribution of subjects based on CYP1A2 activity (17MX+ 17MU)/1 37MX ratio

There was clear evidence of a tri-modal distribution of (17MX +

17MU)/137MX ratio with an apparent anti-mode in the region of 2.5 and 6.5 which was in close agreement to that observed by several studies (Afonina I et al 1997).

The mode corresponding to the rapid enzyme activity was not uniformly distributed and may include both heterozygous and homozygous rapid phenotype. The frequency distribution histogram suggests the possible existence of a second anti- 99 mode (in the region of 6.5) separating the heterozygous and homozygous rapid phenotype.

A total of six different genotypes were found, four associated with rapid phenotype and two with slow phenotype. The most common genotype found was

CYP1A2*1A1*1A with frequencies of 0.825. Followed by alleles CYP1A2*1A1*1C and

CYP1A2*1A1* 1 K with frequency of 0.102 and 0.058 respectively. The

CYP1A2*1CI* 1C and CYP1A2*31*3 genotypes showed significantly the lowest

median values 2.06 and 2.00 respectively (Table 20).

Table 20: (17MX + 17MU)/1 37MX molar ratios in subgroups assigned to CYP1A2 allele combinations Maximum CYP1A2 CYP1A2 N Frequency Median Minimum phenotype alleles Rapid *1A1*1A 461 0.825 7.58 6.08 25.25 Inte rmediate *1A1*1C 57 0.102 4.98 3. 13 6.96 *1A1* 1K 33 0.058 4.87 2.53 6.87 *1Al*3 1 0.002 6.85 6.85 6.85 Slow *1C/*1C 7 0.012 2.06 0.1 2.39 *31*3 1 0.002 2.00 2.00 2.00 Total 560

The resulting urinary (17MX + 17MU)/137MX ratio and genotypic

assignments of slow (SIS homozygotes), intermediate (RIS heterozygotes) and

rapid (R/R homozygotes) are presented in table 21 .. Using Kruskal-Wallis test, the

mean rank of these groups were 4.5, 67.71 and 327.29 respectively, which

represented strong association between CYP 1 A2 genotype and phenotype (X2 =

219.23, df = 2 and P value < 0.0001; 95% CI of differences).

Table 21: (17MX + 17MU)/1 37MX ratios vs. genotypes. Suggested genotypic state: SIS homozygously slow, RIS heterozygotes intermediate, R/R homozygously rapid

SIS homozygotes RlS heterozygotes RlR homozygotes

N 8 91 461 9.3085 Mean 1.8400 5.0830 100

Median 2.0300 4.9800 7.5800 Std. Deviation 0.74310 1.36216 3.37762 Range 2.29 3.95 18.75 Minimum 0.10 2.53 6.08 Maximum 2.39 6.98 25.25

SIS homozygotes slow metabolizers, RlS heterozygotes = intermediate metabolizers, RlR homozygotes = rapid metabolizers

In this study, the phenotype status of CYP1A2 activity in the Emirati population shows the lowest frequency of poor metabolizers with only 1.4% slow phenotype while the percentage in other populations such as Australians, Japanese,

Chinese, Americans and Italian were 5%, 14%, 5%, 12% and 13% respectively

(Figure 29) (Butler et al 1992, Nakajima M et al 1994, Zhou SF et al 2009b).

11'�

10"",

4 ,

2%

0% Au!.lril IIJn� Jupilnc!.c Chlnc!.c Amcriciln!. Ililliiln�

in Figure 29: The frequency of CYP1A2 poor metabolizers among Emiratis 576), Australians comparison with other populations, n = sample size, Emiratis (n = (n = 78), Americans (n = 101), Italians (n = (n = 171), Japanese (n = 250), Chinese 95)

to 81.6% based The degree of phenotype/genotype concordance was equal ate/slow phenotypes), on the current phenotype classification (i.e. rapid/intermedi ele CYP1A2*1A genotypes are where those who were homozygotes for the all were heterozygotes for the wild considered to be a rapid metaboliser, those who metaboliser and those who had no type allele are considered to be intermediated a slow metaboliser. CYP1A2*1A genotype are considered to be 101

CYP1A2 and Degree of Parent's Relationship

Out of8 subjects "homozygote for the mutant alleles", 2 (25%) subjects were children of third degree relationship, 1 (12.5%) subject was their parents from the same tribe and 5 (62.5%) subjects were not related. On the other hand, out of 568 subjects "homozygote or heterozygote for the wild type allele", 183 (32.2%) subjects were children of third degree relationship, 104 (18.3%) subjects were parents from the same tribe and 281 (49.%) subjects were not related (Likelihood Ratio = 0.552, degree of freedom (df) = 2 and P value = 0.759; 95% CI of differences) which was more than 0.05 representing a weak evidence of a relationship or association between genotype and degree of relationship. There was no significant correlation between degree of relationship and genotypes with correlation coefficient 0.028 value (P value = 0.508; 95% CI of differences using Spearman's rho statistical test).

CYP1A2 and "Medical Illness"

With regard to medical illness, it was found that out of 56 cases 1 (1 .8%) subject who had medical illness was from those who were homozygote for the mutant alleles compared to 55 (98.2%) subjects from those who were homozygote or heterozygote for the wild type allele (Likelihood Ratio = 7.296, degree of freedom

(df) = 10 and P value = 0.697; 95% CI of differences) which was more than 0.05 represented a weak evidence of a relationship or association between genotype and medical illness. There was no significant correlation between medical illness and genotypes with correlation coefficient 0.010 value (P value = 0.804; 95% CI of

differences using Spearman's rho statistical test).

Out of 8 subjects "homozygote for the mutant alleles", 1 (12.5%) subject had

Out of medical illness compared to 7 (87.5%) subjects who had no medical illness. allele" 55 (9.9%) 568 subjects "homozygote or heterozygote for the wild type that had no medical subjects had medical illness compared to 513 (90.1 %) subjects 102 illness with OR = 1.306, relative risk for those with medical illness 1.268 and relative risk for those with no medical illness 0.971 (Likelihood Ratio of X2 = 0.058, df = 1 and P value = 0.81; 95% CI of differences).

CYP1A2 and Drug Sensitivity

With regard to drug sensitivity, it was found that out of 21 cases 1 (4.8%) who reported drug sensitivity was from those who were homozygote for the mutant alleles compared to 20 (95.2%) subjects from those who were homozygote or heterozygote for the wild type allele (Likelihood Ratio of X2 = 1.339, degree of freedom (df) = 3 and P value = 0.72; 95% CI of differences) which was more than

0.05 represented a weak evidence of a relationship or association between CYP1A2 genotype and drug sensitivity. There was no significant correlation between drug sensitivity and genotypes with correlation coefficient 0.056 value (P value = 0.179;

95% CI of differences using Spearman's rho statistical test).

Out of 8 subjects "homozygote for the mutant alleles", 1 (12.5%) subject was having drug sensitivity compared to 7 (87.5%) subjects with no drug sensitivity. Out of 568 subjects "homozygote or heterozygote for the wild type allele" 20 (3.5%) subjects had drug sensitivity compared to 548 (96.5%) subjects who had no drug sensitivity with OR = 3.914, relative risk for those with drug sensitivity 3.55 and relative risk for those with no drug sensitivity 0.907 (Likelihood ratio of X2= 1.14, df =

1 and P value = 0.286; 95% CI of differences).

Using Kruskal-Wallis Test, it was found that there was no difference in the

Mean Rank between the groups which represented a lack of effect of CYP1A2 genotype on the Drug sensitivity (X2 = 0.909, df = 3 and P value = 0.823; 95% CI of differences).

CYP1A2 and Genotype Distribution within the UAE Emirates 103

Out of the 8 subjects "homozygote for the mutant alleles" 5 (62.5%) were from AD Emirate, 3 subjects (37.5%) were from Dubai Emirate and no one was found from Northern Emirates. On the other hand, out of 568 subjects "homozygote and heterozygote for the wild type allele" 261 (46%) were from AD Emirate, 108

(19%) were from Dubai Emirate and 199 (35%) were from Northern Emirate.

However, this resulted in significant statistical difference between the cities

(Likelihood ratio of X2 = 7.085, df = 2 and P value = 0.029; 95% CI of differences) with a positive small effect based on the symmetric measures of Phi and Cramer's V values (Phi = 0.09 and Cramer's V= 0.09) with insignificant P value (P value =

0.097; 95% CI of differences).

4.4.2 NAT2

4.4.2.1 NA T2 Genotype

Genetically, we were able to define 570 subjects using PCR-RFLP analysis whereas DNA sequencing has been done for the remaining 6 samples. It was found that out of 576 subjects 446 (77.4%) subjects were carrier of 2 mutant alleles either homozygote or heterozygote, 106 (18.4%) were heterozygote for one mutant allele and 24 (4.2%) homozygous for the wild type allele (Figure 30).

Homozygote Homozygote or Heterozygote Wild Type Heterozygote for 2 mutnat alleles 104

Figure 30: The genotype frequencies- 0.774 of our subjects were homozygote or heterozygote for 2 mutant alleles genotyping, 0.184 were heterozygote and 0.042 were homozygotes for the wild type genotyping

The overall mutant alleles' frequency was 0.866 with allele NA T2 *5 frequency = 0.364, allele NA T2*6 = 0.129, allele NA T2 *7= 0.274 and allele NA T2*14

= 0.099 while the wild type allele frequency was found to be 0.134 (Figure 31).

Figure 31: Allele frequencies- allele NA T2 *5 frequency = 0.364, allele NA T2*6 = 0.129, allele NA T2*7= 0.274 and allele NA T2 *14 =0.099. The wild type allele frequency was found to be 0.134

The different patterns of RFLPs obtained after separate restriction enzyme digestion of the PCR product with Mspl, Fokl, Odel, Kpnl, Taql and BamHI are shown in the following figure 31. The presence of a Kpnl site is indicated by fragments of size 424bp and 135bp. Similarly, the presence of a BamHI site is indicated by the fragments size 515bp and 44bp. In each instance, the presence of

559bp, indicates loss of the cutting site, and is diagnostic for 481 C>T (Kpnl) or

857G>A (BamHI).

After digestion with Taql, site was indicated by fragments of size 226bp and

170bp. Fragments size 142bp and 21 bp also appeared but were uninformative. The presence of 396bp fragment indicates the loss of the polymorphic Taql site and is 105 diagnostic for the 590G>A mutation. After digestion with Mspl, site was indicated by fragments of size 181 bp and 93bp. Fragments size 168bp also appeared but was uninformative. The presence of 274bp fragment indicates the loss of the polymorphic Mspl site and is diagnostic for the 191 G>A mutation.

The presence of Fokl site is indicated by fragments of size 337bp and

105bp. the presence of 442bp fragment indicates the loss of polymorphic Fokl site and is diagnostic for the 282C>T mutation. After digestion with Odel from the first

PCR (442bp), site was indicated by fragment size 221 bp. Fragments size 163bp and

58bp also appeared but were uninformative. The presence of 189bp and 32bp fragments indicates the loss of the polym orphic Odel site and is diagnostic for the

341 T>C mutation. Whereas, the digestion with Odel from the second PCR (559bp), site was indicated by fragment size 124bp. Fragments size 345bp and 90bp also

appeared but were uninformative. The presence of 97bp and 27bp fragments

indicates the loss of the polymorphic Odel site and is diagnostic for the 803A>G

mutation. The presence of wild type (NA T2 *4) allele in subjects phenotyped as rapid

acetylators was inferred by exclusion of each of the 191 G>A, 341 T>C, 590G>A and

857G>A mutations.

The following figure (32) shows the gel documentation of the ONA fragments after

amplification and restriction cleavage. 106

Figure 32: Representative images of the gel documentation of the DNA fragments after amplification and restriction cleavage. bp = base pairs, w = wild type = reference sequence (RS) and m = mutant = sequence variation (SV)

We were able to identify 27 NAT2 diplotypes with the actual genotypes identified in the study group shown in Table 29. The ambiguous diplotypes were clarified completely by haplotype reconstruction with a probability of the reconstructed haplotype pairs of P>O.97. The vast majority of haplotype pairs were assigned with a high degree of confidence (P=1 .00) with two having a probability of

P�O.99 and two others P�O.98. 107 Out of the 576 subjects, 130 (22.6%) subjects were homozygote and heterozygote for the wild type allele. Of whom, 24 (4.2%) subjects were homozygote for the wild type allele, 72 (12.5%) subjects were heterozygote for NA T2 *4/*5 genotype, 22 (3.8%) were heterozygote for NA T2*4/*6 genotype, 7 (1 .2%) were heterozygote for NA T2*41*7 genotype and 5 (0.9%) were heterozygote for

NA T2 *41*14 genotype. Whereas, 446 (77.4%) subjects were homozygote and heterozygote for the mutant alleles. Of whom, 36 (6.2%) subjects were homozygote for alleles NA T2*5 cluster, 78 (13.5%) subjects were heterozygote for NA T2 *5B/*6A genotype, 177 (30.7%) subjects were heterozygote for NA T2*5/*7 clusters, 20

(3.5%) subjects were heterozygote for NA T2 *5/* 14 clusters, 5 (0.9%) subjects were homozygote for NA T2*6 cluster, 34 (5.9%) subjects were heterozygote for

NA T2*6A/*7B genotype, 5 (0.9%) subjects were heterozygote for NA T2 *6A/*14B genotype, 14 (2.4%) subjects were homozygote for NA T2*7B/*7B genotype, 69

(11 .9%) subjects were heterozygote for NA T2*71*14 clusters and 7 (1.2%) subjects were homozygote for NA T2*14A/*14B genotype (Table 22).

Table 22: NAT2 diplotypes and genotypes in the study group. Comparison between genotypes reconstructed by PHASE and actual genotypes obtained by gene mapping

Observed No. Actual PHASE Probability diplotype* genotypes reconstruction

0000000 24 *41*4 *4/*4 1.00 001 1000 4 *4/*5A *4/*5A 1.00 001 1010 52 *4/*58 *4/*58 0.98 001001 0 6 *4/*5C *4/*5C 0.98 0010000 11 *41*50 *41*50 1.00 0100100 19 *4/*6A *4J*6A 1.00 0000100 5 *41*68 *41*68 1.00 0100001 8 *4/*78 *41*78 1.00 1000000 6 *41*14A *41*14A 1.00 0022000 2 *5A/*5A *5A/*5A 1.00 0022010 9 *5A/*58 *5A/*58 1.00 01 11001 18 5A/*78 *5A/*78 1.00 1011000 4 *5A/*1 4A *5A/*1 4A 0.99 0022020 17 *581*58 *58/*58 1.00 108

0021020 5 *58/*5C *58/*5C 1.00 0222220 3 *5U/*5U *5U/*5U 1.00 0111110 78 *58/*6A *58/*6A 1.00 011101 1 153 *581*78 *58/*78 1.00 1111010 1 6 *58/* 148 *58/*148 0.99 0200200 4 *6AJ*6A *6AJ*6A 1.00 0200220 1 *6C/*6C *6C/*6C 1.00 0200101 34 *6A/*78 *6A/*78 1.00 1200100 5 *6A/*148 *6A/*148 1.00 0200002 14 *78/*78 *78/*78 1.00 1100001 9 *78/* 14A *78/*14A 1.00 1200001 61 *78/* 148 *78/*148 1.00 2100000 7 *14AJ* 148 *14AJ* 148 1.00 Total 576 * Observed diplotypes are shown as the number of mutations identified in each individual, 0: homozygous reference, 1: heterozygous, 2: homozygous variant; the SNP order is 191, 282, 341, 481 , 590, 803 and 857. Ambiguous diplotypes with at least two heterozygous loci are highlighted.

The frequencies of the various haplotypes identified among Emiratis determined from PCR-RFLP analysis of genomic DNA are shown in Table 23. The most common haplotypes by far among our population sample belonged to the *58 gene cluster and occurred with a frequency of 0.301, which is almost three times the frequency of the wild type allele (NA T2*4). Furthermore, a second slow allele belonging to the NA T2*78, was also more common than allele NA T2 *4, occurring with a frequency of 0.274.

Examining the deviation from Hardy-Weinberg Equilibrium, it was also found that the observed genotype frequencies were different from the expected one.

However, the apparent numbers of alleles NA T2*7 and NA T2 *14 were not in strict accordance with the Hardy-Weinberg Equilibrium. For alleles NA T2*5 and NA T2*6 the data obey the equilibrium since the result was statistically insignificant (variant allele frequency for allele NA T2*5 = 0.55, P value = 0.25; 95% CI of differences and for allele NA T2*6 = 0.31, P value = 0.98; 95% CI of diffe rences) (table 23).

Table 23: The haplotype frequencies of NAT2 109

Haplotype Frequency Percentage Hardy-Weinberg Equilibrium GCTCGAG *4 154 13.4% GCCTGAG *5A 45 3.9% GCCTGGG *5B 347 30.1% Var. allele freq.= 0.55, P=0.25 GCCCGGG *5C 11 0.95% GCCCGAG *5D 10 0.86% GTCTAGG *5U 6 0.52% GTICAAG *6A 143 12.4% Var. allele freq.= 0.31, P=0.98 GCTCAAG *6B 4 0.34% GTICAGG *6C 2 0.17% GTICGAA *7B 316 27.4% Var. allele freq.= 0.39, P<0.001 ACTCGAG *14A 25 2.2% ATICGAG *14B 89 7.76% Var. allele freq.= 0.26, P<0.001 Var. allele freq.= Variant allele frequency

Based on epidemiological studies, the frequency of NAT2 slow acetylation alleles in the Emirati population shows the highest frequency with 86.6%. However, in Egyptian, Indian, Omani and Jordanian populations, the frequency of the slow acetylation alleles ranged between (74% - 79%) while in the Moroccan population it was 84% (Meyer UA and Zanger UM 1997, Guaoua S et al 2014). Moreover, in

American, German, Spanish, Argentinian and Saudi Arabian populations the frequency was between (72%-73%), whereas in African and Southern Brazil populations it reached 40%, in Senegalese population 60% and in Tunisian population 69%. These populations have high frequencies of mutant allele NA T2*5 and NA T2*6 and low frequencies of mutant allele NA T2 *7. In Asian populations, such as Japanese, Chinese, Korean, and Thai, the frequency of the slow acetylation alleles ranged from 10 to 30% with high frequency of mutant allele NA T2 *7 and low frequency of mutant allele NA T2*5 ( Figure 33) (Meyer UA and Zanger UM 1997,

Guaoua S et al 2014). 110

100 90 80 70 60 50 1- - 40 30 20 10 o � r

Figure 33: The frequency of NA T2 slow acetylation alleles among Emiratis in comparison with other populations, n = sample size, Emiratis (n = 576), Asians "Japanese (n = 79), Koreans (n = 288), Chinese (n = 120), Thai (n = 235)", Africans (n = 97), Americans (n = 387), Tunisians (n = 100), Indians (n = 250), Egyptians (n = 200), Germans (n = 844), Omanis (n = 127), Argentineans (n = 185), Spaniards (n = 258), Saudi Arabians (n = 200), Moroccans (n = 163), Jordanians (n = 150)

Identification of Known Slow Alleles Subtype

DNA sequencing was performed for 20 samples randomly selected to confirm the accuracy of our PCR�RFLP results. Nevertheless, DNA sequencing was also performed for the remaining 6 samples that had not been clearly defined using

PCR�RFLP.

Table 24 provided more detailed data of the 6 samples that had been analysed using DNA sequencing. Whereas, figure 34 illustrated DNA chromatogram that showed 510T>C variant of sample 3 DNA with well�resolved peaks and no ambiguities. 111

Table 24: Details of the . results of the 6 samples that has been analysed using DNA sequencing

Sample Variants Allele1 Allele2 Assigned Number Genotype Sample 282C>T 590G>A 282C>T 590G>A 282C>T 590G>A Homozygote 1 341T>C 803G>A 341T>C 803G>A 341T>C 803G>A NA T2'5U1*5U 481C>T 481 C>T 481C>T

Sample 341T>C 803G>A 341T>C 803G>A 34 1T>C 803G>A Homozygote 2 481C>T 481 C>T 48 1C>T NA T2'5B1*5B

Sample 282C>T 590G>A 282C>T 282C>T 590G>A Heterozygote 3 S10T>C 803G>A 590G>A S10T>C 803G>A NA T2*6CI*6C? 77SA>C 803G>A 77SA>C

Sample 282C>T 803G>A 282C>T 803G>A 282C>T 803G>A Heterozygote 4 341T>C 77SA>C 341T>C 34 1T>C 77SA>C NA T2*5UI*5U? 481C>T 733A>C 481 C>T 481 C>T 733A>C 590G>A 590G>A 590G>A

Sample 34 1T>C 803G>A 34 1T>C 803G>A 34 1T>C 803G>A Homozygote 5 481C>T 481 C>T 48 1C>T NA T2*5B1*5B

Sample 34 1T>C 803G>A 34 1T>C 803G>A 341 T>C 803G>A Homozygote 6 590G>A 481 C>T 590G>A 481 C>T 590G>A 481 C>T NA T2*5U1*5U 282C>T 282C>T 282C>T The new variants are highlighted

T C : C ..\ 7 C T C

Nucleotides peaks At pos 'on 510, Y representstwo peaks of m ThYmine & (e) C) osine

Figure 34: An example of DNA chromatogram of T-51 OC variant of sample 3 DNA

NA T2*58

Out of these 6 samples, two were homozygote for NA T2 *5B, an allotypic variant of NA T2 *5, that is characterized by presence of 3 mutations,

341 T>C,481 C>T and 803G>A. It differs from NA T2*5 only by the presence of the

341 T>C and 803G>A mutations and is indistinguishable from NA T2*4 "wild type 112 allele" when the criterion for identification of NA T2 *4 IS based only upon exclusion of the common NA T2 slow alleles. Allele carrying 34 1 T>C and 481 C>T mutations were presumed to be NA T2*5A in the absence of 803G>A mutation, while alleles carrying

282C>T, 341T>C, 481 C>T and 803A>G mutations were presumed to be NA T2*5G.

Accordingly, we confirmed the presence of these 3 mutations only by DNA sequencing of 2 samples.

NA T2 *5U

Out of these 6 samples, one sample was heterozygote and two were homozygote for NA T2*5U, an allotypic variant of NA T2 *5, that is characterized by presence of 5 mutations, 282C>T, 341T>C, 481C>T, 590G>A and 803>A. It differs from NA T2*5 only by the presence of the 282C> T, 34 1 T>C, 590G>A and 803G>A mutations. Alleles carrying 341T>C, 481 C>T and 803G>A mutations only were presumed to be NA T2*58 in the absence of 282C>T and 590G>A mutations, while allele carrying 282C>T, 341T>C, 481C>T and 803G>A mutations were presumed to be NA T2*5G in the absence of 590G>A mutation. Accordingly, we confirmed the presence of these 5 mutations by DNA sequencing of 3 samples.

NA T2*6C

Out of these 6 samples, one sample was heterozygote for NA T2 *6C, an allotypic variant of NA T2*6, that is characterized by presence of 3 mutations,

from NA T2 *6 only by the presence of the 282C>T , 590G>A and 803G>A. It differs

282C>T and 803G>A mutations. Alleles carrying 590G>A, 282C>T and 111T>C and mutations were presumed to be NA T2*60, while allele carrying 590G>A of 282C>T 803G>A mutations were presumed to be NA T2 *6F in the absence only by DNA mutation. Accordingly, we confirmed the presence of these 3 mutations

sequencing of one sample. 113 Unreported Variants and Unknown Slow Alleles Subtype

After the confirmation of the presence of two samples that were homozygote for NA T2*5B, two samples were homozygote for NA T2*5U, one sample was heterozygote for NA T2*6CINat2*6? (Unreported) and one sample who was heterozygote for NA T2*5UINA T2*5? (Unreported). Three new variants were confirmed using DNA sequencing with repetitions. These variants have been checked in different databases like Ensembl, ExAC and GlaxC database and were found that they were not reported before. The new variants were "510T>C, 775A>C and 733A>C". They were presented as heterozygote in NA T2*5 and NA T2*6 clusters and related to nucleotides changes in the protein coding regone.

4.4.2.2 Caffeine Metabolites Ratio and NAT2 Phenotype Status

Since other studies had used (AFMU/1 MX) molar ratio, in this study, we used similar measures of calculation and comparison. Caffeine phenotype categories were defined as slow, intermediate or rapid activators based on the distribution of these values with cut points at 0.5 and 2 (see aforementioned Table

10) (Grant OM et al 1984 and Woolhouse N et al 1997).

The ratio of AFMU/1 MX was calculated to determine the phenotype status of

NAT2 activity in each subject. Out of 576 subjects, 555 showed positive results whereas the data from the remaining 21 were missing. From those 555 subjects,

436 (78.5%) were slow acetylators for NAT2 activity "ratio < 0.5", 106 (19.1%) were intermediate "ratio between 0.5 _2" and 13 (2.4%) subjects were rapid acetylators for this enzyme "ratio > 2" (Table 25).

Table 25: The classification and the results of the acetylator status of NAT2 activity and its sub-classification based on (AFMU/1 MX) ratio

AFMU/1 MX ratio Acetylator status No. of subjects Percentage

0-0.5 Slow NAT2 acetylator 436 78.5% 114

0.5-2 Intermediate NAT2 acetylator 106 19.1% 2- above Rapid NA T2 acetylator 13 2.4%

The frequency distribution histogram of urinary AFMU/1 MX molar concentration ratio for 555 UAE nationals was non-normally distributed (Median =

0.2535, Skewness = 3.633 with Std error = 0.104, Kurtosis = 18.418 with Std error =

0.207) (Figure 35).

lllCl

110

IOU

liD

( .n

lin 10 I (1 • - • - - - ...... ,..., ""I '-'> I ClO eJ' - - � -< ..,., <=> <=> e- o "'" ." 0 r-:i 0 ...I ::l -I .... -I (AMF U/l.M)()ro Uo

Figure 35: The distribution of subjects based on NAT2 activity (AMFU/1 MX) ratio

There was a clear evidence of a bi-modal distribution of AFMU/1 MX ratio with an apparent anti-mode in the region of 0.5. The mode corresponding to the slow acetylator phenotype was uniformly distributed and was assumed to be made up of individuals who possess various combinations of several diffe rent alleles, known to determine slow acetylation.

The mode corresponding to the rapid acetylator phenotype was not uniformly distributed and is assumed to include both heterozygous and homozygous rapid acetylators. The frequency distribution histogram suggests the possible existence of a second anti-mode (in the region of 2) that separates the heterozygous and homozygous rapid acetylators.

Table 26 and figure 36 shows the acetylation capacity of the 25 subgroups assigned to NAT2 allele combinations. The NA T2*6A/*6A, NA T2*6A1*7B, 115 NA T2 *7BI"7B, NA T2 *5A/*5B and NA T2*5A/*5A genotypes showed significantly the lowest median values 0.006, 0.018, 0.029, 0.095 and 0.132 respectively. Nine different genotypes associated with rapid acetylation and sixteen with slow acetylation, the most com mon genotypes found were NA T2*5B/*7B heterozygotes,

NA T2* 5BI*6A heterozygotes, NA T2*7B/*14B heterozygotes and NA T2 *4/*5B heterozygotes with frequencies of 0.255, 0.135, 0.105 and 0.09, respectively.

Table 26: AFMU/1X molar ratios in subgroups assigned to NAT2 allele combinations

Phenotype NAT2 alleles or N Median Minimum Maximum pos ible allele combination Rapid *4/*4 13 2.5430 2.0634 4.3321 Intemlediate *4/*5A 4 0.5788 0.5249 0.7992 *4/*5B 52 0.7955 0.5200 1.8045 *4/*5C 6 0.9055 0.61 02 1.6078 *4/*5D 10 0.8665 0.6190 1.7049 *4/*6A 18 0.8435 0.4569 1.7478 *4/*6B 4 1.6562 1.6078 1.6790 *4/*7B 7 0.7326 0.5633 1.0496 *4/* 14A 5 0.7645 0.5382 1.4808 low *5A/*5A 2 0.1 323 0.1210 0.1436 *5A/*5B 9 0.0956 0.0281 0.5269 5A/*7B 24 0.2670 0.0979 0.5445 *5A/* 14A 4 0.4151 0.0815 0.4649 *5B/*5B 17 0.2455 0.0087 0.5360 *5B/*5C 3 0.2422 0. 1321 0.2500 *5B/*6A 78 0.2058 0.0146 0.5488 *5B/*7B 149 0.2152 0.0120 0.6400 *5B/* 14B 16 0.3586 0.01 42 0.5275 *6A/*6A 4 0.0061 0.0021 0. 1497 *6A/*7B 34 0.0188 0.0034 0.1 996 *6A/* 14B 5 0.3803 0.0356 0.5125 *7B/*7B 14 0.0298 0.005 1 0.1 489 *7B/* 14A 9 0.2453 0.1 726 0.4808 *7B/* 14B 61 0.3361 0.0134 0.5787 * 1 4A/* 14B 7 0.2037 0.0147 0.5116 Total 555 116

o � - eo<: 2 -. %

:...J

c;:-' .

. . (I, .

' ( � Co < )' 1 "P s Figure 36: Comparison of NAT2 phenotype and genotype in 555 subjects. It illustrates the acetylation capacity of the 25 subgroups assigned to NAT2 allele combinations. Those who are homozygotes for NAT2*4 alleles show the highest AFMU/1 MX ratios, those who are heterozygotes for NAT2*4 allele show intermediate AFMU/1 MX ratios and those who are homozygotes or heterozygotes for the derived alleles show the slowest AFMU/1 MX ratios

The resulting urinary AFMU/1 X ratios and genotypic assignments of slow

(SIS homozygotes), intermediate (RIS heterozygotes) and rapid acetylators (RIR

homozygotes) are presented in Table 27. Using Kruskal-Wallis test, the mean rank

of these groups were 217.8, 487.2 and 549 respectively, which represented strong

association between NAT2 genotype and phenotype (X2 = 282.01 , df = 2 and P

value < 0.0001; 95% CI of differences).

Table 27: AFMU/1X ratios vs. genotypes, based on the last update of NAT nomenclature scheme published by The Arylamine N-acetyltransferase Gene Nomenclature Committee according to consensus guidelines (Hein OW et al 2008). Genotypic acetylator state: SIS homozygously slow, RIS heterozygotes, RlR homozygously rapid

SIS RlS RlR homozygotes heterozygotes homozygotes N 436 106 13 Mean 0.25 0.89 2.76 SO 0.16 0.35 2.62 Median 0.22 0.79 2.54 Range 0.53 1.28 2.27 Minimum 0.01 0.52 2.06 Maximum 0.53 1.8 4.33 117 SIS homozygotes slow acetylators, RlS heterozygotes = intermediate acety lators, R/R homozygotes = rapid acetylators

In this study, the phenotype status of NAT2 activity in the Emirati population shows the highest frequency of poor metabolizers with 78.5% slow phenotype while the percentage in other populations such as Americans, Turkish, Egyptians and

Saudi Arabians were 58.1 %, 57.4%, 60.5% and 72.3% respectively (Figure 37)

(Hamdy et al 2003, Ojordjevic N et al 2012).

!)O

so

70

GO

50

40

30

20

10

o Americans Turklsl1 EgyptlOJns SaudiAr ablal1s Emlratis

Figure 37: The frequency NAT2 poor metabolizers among Emiratis in comparison with other populations, n = sample size, Emiratis (n = 576), Americans (n = 255), Turkish (n = 113), Egyptians (n = 200), Saudi Arabians (n = 200)

The degree of phenotype/genotype concordance was calculated by measuring the agreement between the genotype and phenotype methods based on the traditional phenotype classification (i.e. rapid/slow phenotypes), where non containing allele 4 genotypes, are considered to be a slow phenotype, and was equal to 96.2%.

NA T2 and Degree of Parent's Relationship

Out of 446 subjects "homozygote or heterozygote for the mutant alleles", 141

(31.6%) subjects were children of third degree relatives, 84 (18.8%) subjects had parents from the same tribe and 221 (49.5%) subjects were not related. On the other hand, out of 130 subjects of the "homozygote or heterozygote for the wild type 118 allele", 44 (33.8%) subjects were children of third degree relatives, 21 (16.2%) subjects had parents were from the same tribe and 65 (50%) subjects were not related (Pearson Chi-Square = 0.567, degree of freedom (df) = 2 and P value =

0.753; 95% CI of differences) which was more than 0.05 representing a weak evidence of a relationship or association between genotype and degree of relationship. There was no significant correlation between degree of relationship and genotypes with correlation coefficient 0.014 value (P value = 0.729; 95% CI of differences using Spearman's rho statistical test).

In addition, it was found that 78.1 % of the subjects from those whose parents were relatives were homozygote or heterozygote for the mutant alleles whereas,

77.4% from those whose parents were not relatives were homozygote or heterozygote for the wild type allele with OR = 1.04 1 , relative risk for those whose parents were relatives was 1.02 and relative risk for those whose parents were not relatives was 0.98 (X2 = 0.040, df = 1 and P value = 0.841; 95% CI of differences).

Using Kruskal-Wallis Test, out 576 subjects, 185 subjects were classified as children with third degree relationship, 105 subjects were classified as relatives from the same tribe and 286 subjects with no relationship. There was no difference in the

Mean Rank between the 3 groups which represented no effect on the NA T2 genotype (X2 = 0.566, df = 2 and P value = 0.753; 95% CI of differences).

NAT2 and "Medical Illness"

To determ ine whether medical illness was correlated with genotypes, we found out of 57 cases, 40 (70.2%) who had medical illness were from those who were homozygote or heterozygote for the mutant alleles compared to 17 (29.8%) from those who were homozygote or heterozygote for the wild type allele (Likelihood

Ratio = 7.488, degree of freedom (df) = 10 and P value = 0.679; 95% CI of

differences) which was more than 0.05 representing a weak evidence of a 119 relatIonship or association between genotype and medical illness. There was no sIgnIficant correlation between medical illness and genotypes with correlation coefficient of 0.007 (P value = 0.957; 95% CI of differences using Spearman's rho statIstical test).

Out of 446 subjects "homozygote or heterozygote for the mutant alleles" , 40

(8.93%) subjects had medical illness compared to 406 (91 .07%) subjects were with no medical illness. Out of 130 subjects "homozygote or heterozygote for the wild type allele" 17 (13.28%) subjects were having medical illness compared to 113

(86.72%) who had no medical illness with OR = 0.64, relative risk for those with medical illness 0.672 and relative risk for those with no medical illness 1.05 (X2 =

2.115, df = 1 and P value = 0.146; 95% CI of differences).

Using Kruskal-Wallis Test, it was found that there were no differences in the mean rank between the groups which represented no effect of NAT2 genotype on

PMH (X2 = 4.909, df = 10 and P value = 0.897; 95% CI of differences).

NA T2and Drug Sensitivity

With regard to drug sensitivity, it was found that out of 21 cases 17 (81 %) who had drug sensitivity were from those who were homozygote or heterozygote for the mutant alleles compared to 4 (19%) from those who were homozygote or heterozygote for the wild type allele (Likelihood Ratio = 1.840, degree of freedom

(df) = 3 and P value = 0.606; 95% CI of differences) which was more than 0.05 representing a weak evidence of a relationship or association between NA T2 genotype and drug sensitivity. There was no significant correlation between drug sensitivity and genotypes with correlation coefficient of 0.022 (P value = 0.925; 95%

CI of differences using Spearman's rho statistical test). 120

Out of 446 subjects "homozygote or heterozygote for the mutant alleles", 17

(3 79%) subjects had drug sensitivity compared to 431 (96.21 %) subjects with no drug sensitivity. Out of 128 subjects "homozygote or heterozygote for the wild type allele" 4 (3.1 3%) subjects were having drug sensitivity compared to 124 (96.88%) who had no drug sensitivity with OR = 1.223, relative risk for those with drug

sensitivity 1.214 and relative risk for those with no drug sensitivity 0.993 (Likelihood

ratio = 0.132, df = 1 and P value = 0.717; 95% CI of differences).

Using Kruskal-Wallis Test, it was found that no differences in the Mean Rank

between the groups which represented no effect of NA T2 genotype on the Drug

sensitivity (X2 = 1.658, df = 3 and P value = 0.646; 95% CI of differences).

NA T2 and Genotype distribution within the UAE Emirates

Out of the 446 subjects who were homozygote or heterozygote for the

mutant alleles 210 (46.69%) were from AD emirate, 90 subjects (20.09%) were from

Dubai emirate and 146 (33.04%) were from Northern Emirates with no significant

statistical difference between the cities (X2 = 2.247, df = 2 and P value = 0.325;

95% CI of differences). 121 Chapter 5: Discussions

This study of 576 subjects of Emiratis is considered to be the first complete and comprehens ive report on CYP1A2 and NA T2 phenotyping and genotyping on UAE population in which all known or common mutations or polymorphisms of these

genes have been fully and thoroughly tested using HPLC and TaqMan Real Time

PCR techniques for CYP1A2 gene and HPLC, PCR-RFLP techniques and DNA

sequencing for NA T2 gene.

The distribution of samples were based on the distribution of population between emirates which was again based on the data provided by UAE National

Bureau of Statistics of Emiratis which represented proper sample distribution all over

UAE.

In the past, acetylator phenotypes were dete rmined using different substances. Traditionally, isoniazid has often been used for acetylator phenotype determination. However, isoniazid undergoes complex metabolism and requires serial blood sampling and would not be convenient to use as a screening tool. Other substances containing sulfonamide like sulfadimidine, sulfapyridine, sulfasalazine or sulfamethazine, and based on the classical Bratton-Marshall procedures, which involve the administration of a single oral dose and collection of multiple samples or a single sample of serum or urine, have been used as well. Dapsone has also been

used with a single sample of plasma being collected 2-72 hours after administration.

The ratio of N-acetylprocainamide to procainamide in plasma has been used to

determine acetylator phenotype. In the present study, caffeine was used because it

is ubiquitous and present in popular drinks like colas, coffees and teas and can be

used in school going children. It is also easier to collect urine samples than blood

samples. In this study, urine samples were co llected two hours after a drink of cola

or coffee. 122

5.1 CYP1A2

The variability in liver expression of the CYP1A2 gene reached almost 40%

and 60% variability in caffeine metabolism; the most often used probe drug for

CYP1A2 (Gunes A and Dahl ML. 2008). Only few variants explain the phenotypic variability in CYP1A2 gene expression, contrarily, it was much more with other drug­

metabolizing CYPs (Ghotbi R et al 2007). Ghotbi R et al in 2007 observed a very

low frequency of the coding sequence variants in White and Asian populations

(Ghotbi R et al 2007). Browning et al in 2010 examined CYP1A2 variation in

Ethiopians, suggested that because of the overall greater incidence of variation

there could be some individuals freed from any CYP1A2 activity in that population

(Browning SL et al 2010).

Generally, it has been discussed previously that the exact positioning of the

cut-off point (anti mode) between phenotypically slow and rapid activities seems

laboratory-dependent and attributed to differences in chromatographic conditions

(Bolt HM et al 2005). In this particular population sample, the frequency distribution

of the (17MX+1 7MU)/1 37MX ratio is clear trimodal.

CYP1A2*1 A has been the reference (or "wild type") allele for the respective

gene that associated with rapid phenotype status. The other variant alleles have

been classified from this by one or more single nucleotide polymorphisms (SNPs).

Based on many studies in many ethnic groups, allele *1A was the most common

allele (Zhou SF et al 2009b, Browning SL et al 2010). Mutant alleles CYP1A2*1C

"G-3860A (rs2069514)", CYP1A2*1K "T-739G (rs2069526), C-729T (rs12720461)

and C-163A (rs762551)", CYP1A2*3 "21 16G>A and 5347T>C (rs56276455)",

CYP1A2*4 "2499A>T (rs72547516)" and CYP1A2*6 "5090C>T (rs28399424)" had

low catalytic activity associated with the slow phenotype (Zhou SF et al 2009a, 123

Browning SL et al 2010) and were less common than the wild type allele (Chevalier o et al 2001, Zhou H et al 2004).

CYP1A2 genotypes have been classified into three different phenotypes:

"slow" (two slow alleles), "intermediate" (1 slow and 1 rapid allele), and "rapid" (2 rapid alleles). In our study we found that 1.4% of our subjects were homozygote for mutant alleles, 16.1 % were heterozygote and 82.5% were homozygote for the wild type allele.

As a matter of fact, some reports suggest that many influential factors may play an important role in correlating CYP1 A2 genotype to CYP1 A2 phenotype. For instance, the drugs which cause induction or inhibition of CYP1A2 enzyme may affect the correlation between CYP1A2 genotype and phenotype. Similarly, the environmental factors such as cigarette smoking can affect CYP1A2 enzymatic activity as well. With regard to our study, all of the participants were non-smokers and the majority of them were aged between 16-19 years of age. Additionally, 90% of them were with no medical illness and not on any concomitant drugs. Thus, we found the high degree of concordance between phenotype and genotypes of

CYP1A2 gene. Interestingly, we found that those with mutant alleles have shown slow phenotype status. Furthermore, those who were homozygote for alleles

CYP1A2*1C and CYP1A2*3 were associated with the slowest enzyme activity.

Hence, when Hardy-Weinberg equation was applied, the apparent numbers of homozygous and heterozygous slow activity were not in strict accordance with the

Hardy-Weinberg equilibrium which is a clear indication that individual genotypes cannot be predicted. For allele CYP1A2*1C the result was inconsistent with Hardy­

Weinberg Equilibrium which may be due to an unexpectedly large number of

CYP1A2*1 C homozygotes. For allele CYP1A2*1 K the result was consistent with

Hardy-Weinberg Equilibrium. This finding may be due to the evolutionary influences, 124 like mate choice, mutation, selection, genetic drift, gene flow and meiotic drive as one or more of these influences are typically present in real populations. The level of consanguinity in the UAE is very high with more than 50% of marriages being consanguineous. Indeed, about 50% of the sample group reported consanguinity of their parents. This clearly influences the genotype frequencies and their deviation from Hardy-Weinberg.

Our study samples included few numbers with PMH and drug sensitivity and when tested for their relation and effect on genotype we found few significant association. Although, we found a clear associated of CYP1 A2 polymorphism and epilepsy with strong statistical evidence of correlation compared to wild type carriers however, the low number of our subjects with epilepsy could be a concern and the need for another study in this regard is important.

Nevertheless, we found the results were statistically significant and differences have been found between cities in regards to genotype and alleles distribution between Emirates. We found CYP1A2 polymorphisms in AD and Dubai

Emirates whereas they were absent in Northern Emirates.

5.2 NA T2

In this study, we use the ratio of AFMU/1 MX to determine the acetylation status or NA T2 activity. Phenotypically, the differences in NAT2 activity (as measured by AFMU/1 MX ratio) between variable populations have been documented and it was markedly high in Koreans compared to Swedes, and this may be due to a higher proportion of the NA T2*4 rapid allele in Koreans and the higher frequency of slow acetylator genotype in Swedes (Djordjevic N et al 2012). In other studies, 58. 1 % of Americans, 57.4% of Turkish, 60.50% of Egyptians and

72.3% of Saudi Arabians were of slow acetylator phenotype. In our study, NAT2 activity showed a high percentage of slow acetylators (78.5%). 125 Generally, it has been discussed previously that the exact positioning of the cut-off point (antimode) between phenotypically slow and rapid acetylators seems laboratory -dependent and attributed to diffe rences in chromatographic conditions (Bolt HM et al 2005). In this particular population sample, the frequency distribution of the AFMU/1 MX ratio is clearly trimodal.

Some studies have reported good correlation between acetylator phenotype measured by caffeine metabolite ratio and NA T2 genotype (Jetter A et al 2004, Rihs

HP et al 2007); contrarily other studies have showed no correlation (Cascorbi I et al

1995, O'Neil WM et al 1997, Zhao B et al 2000, Wolkenstein P et al 2000, Bolt HM et al 2005, Ojordjevic N et al 201 1). In our study we found a strong statistical evidence of correlation between acetylation-phenotypes and genotypes of NA T2 gene.

The high overall degree of concordance between NA T2 genotype and phenotype thus confirms the validity of genotyping tests to predict NAT2 phenotypes within the UAE population. Genotyping offers several advantages over phenotyping methods; it is a simple and a reliable procedure, obviating the need for the use of probe drugs and is not subject to physiologically or pathologically determined variation. In this particular population sample, the frequency distribution of the

AFMU/1 MX ratio is clear trimodal since those who were identified as NA T2 *4/*4

(wild type) homozygotes, proved to have the highest ratios. Those who were heterozygotes proved to have intermediate ratios and those who were homozygotes for the mutant alleles proved to have the slowest ratios thus providing a strong evidence of gene dosage effect.

Studies on NAT2 are obstructed by issues with unclear haplotype assignment as unphased genotypes of the NAT2 gene SNPs resulted in more than one possible haplotype pair particularly if more than one of these SNPs is 126 heterozygous. Because of this, PHASE v2.1.1 program was used to enable a rapid and accurate haplotype prediction. However, higher precision of the estimates and more unambiguous information may be obtained by increasing the sample size

(Aqundez JA et al 2008 and Selinski Set al 201 3a).

NA T2 is a polymorphic gene with around 110 NA T2 alleles being assigned official symbols by the Arylamine N-acetyltransferase Gene Nomenclature

Committee, according to consensus guidelines (Hein OW et al 2008). Based on many studies in many ethnic groups, haplotype NA T2*4 carries the wild type allele at all the variable sites (polymorph isms) reported in the NA T2 literature that associated with rapid acetylator phenotype and is not the most common allele (Hein

OW et al 1994, Hein et al 1995, O'Neil WM et al 2000). Mutant allele at positions

191, 341, 560 and 857 that represent mutant allele NA T2 *14, NA T2*5, NA T2 *6 and

NA T2*7 respectively, have low catalytic activity associated with the slow acetylator phenotype and are more common than the wild type allele (NA T2*4) (Hein OW et al

1994, Hein et al 1995, O'Neil WM et al 2000). In our study, the frequency of the wild type allele (NA T2*4) among Emiratis was markedly low (0.134) which is similar to many ethnic populations including Caucasians, Egyptians, Omanis, Saudi Arabians,

Indians and Moroccans. However, it is markedly considerably lower in comparison to Asians and Hispanics.

The NA T2gene has a high frequency of functional variation, distinguishing amongst populations that are ethnically different, and has high levels of haplotype variation (Patin E et al 2006a, Mortensen HM et al 201 1). The distribution of polymorphisms in our studied population showed high percentage of those with mutant alleles either homozygote or heterozygote (77.4%).

NA T2 genotypes have been classified into three different phenotypes: "slow acetylator" (two slow alleles), "intermediate acetylator" (1 slow and 1 rapid allele), 127 and "rapid acetylator" (2 rapid alleles) (Stanley LA and Sim E 2008). Some papers used to reportra pid as any genotype containing allele NA T2*4 and slow as any non­ carriers of allele NA T2*4 acetylators (Soejima M et al 2007). In addition, within the slow acetylator genotype group there is heterogeneity in phenotype due to variation in enzyme activity conferred by different alleles (Hein OW et al 1995, Cascorbi I et al

1999, Hein OW 2009, Ruiz JO et al 2012) which may affect the ability to identify significant associations (Selinski Set al 2013b).

Cartwright RA et al (1982) divided the ratio that was used to measure the phenotype status into 4 groups, (0.3 and greater) corresponded to rapid acetylators, and the other three (0.01-0.09; 0.1-0.19; and 0.2-0.29) corresponded to different levels of slow acetylator phenotype. Urinary bladder cancer risk was markedly elevated as NAT2 phenotype reduced, particularly with the slowest NAT2 phenotype

(Cartwright RA et al 1982). Similar conclusion has been reported by different studies

(Brockmoller Jet al 1996, Okkels H et al 1997, Filiadis IF et aI 1999).

Interestingly, we observed that the NA T2 *6A/*6A , NA T2 *6A1*7B,

NA T2*7BI*7B, NA T2*5A1*5B and NA T2*5A1*5A genotypes were having the lowest enzyme activity, respectively. This result was supported by Selinski et al 2013b who identified the ultra-slow genotypes. This 'ultra-slow' genotype is defined by a combination of NA T2*6A1*6A, NA T2*6A1*7B, NA T2 *7B/*7B haplotype pairs (Ruiz JO et al 2012, Selinski et al 2013b). This provided a clear picture that NA T2 slow acetylator phenotype was not homogeneous, but rather multiple slow acetylator phenotypes that exist resulting from different SNPs and mechanisms.

In addition, a lot of the therapeutic agents have been found to be polymorphically acetylated in humans due to the genetic polymorphism in N­ acetyltransferase activity like hydralazine, phenelzine, phenylenediamine, sulphamethazine, isoniazid, sulphasalazine, amonafide, endralazine, procainamide, 128

a number of sulphonamides, nitrazepam and dapsone. Although, the possibility of failed or less effective clinical response as a result of acetylation polyphorphism is very low because of the wide therapeutic window of the drugs as acetylation is a

minor metabolic pathway (Yamasaki Y et al 2008, Ma JJ et al 2009) (Kuhn UO et al

2010). In another study on Japanese patients who were treated with co-trimoxazole found individuals to have higher risk of adverse events with slow acetylator status

(NA T2 genotypes *6A1*6A, *6A/*78, *781* 78) compared to rapid acetylators (NA T2

genotypes *41*4, *41*58, *41*5E, *4/*6A, *4/*78) (Soejima M et al 2007).

NA T2 alleles containing the 191 G>A (rs1801279), 341 T>C (rs1801280),

590G>A (rs1799930), and/or 857G>A (rs1799931) missense substitutions are

associated with slow acetylator phenotypes. Striking ethnic differences in the

frequencies of these missense substitutions (Grant OM et al 1997) play an important

role in ethnic differences in frequency of slow acetylator alleles and phenotypes

(Cascorbi I and Roots I 1999). For example, the 191 G>A substitution commonly

represents the NA T2*14 gene cluster that is available in greater extent in African

Americans and native Africans, but it is virtually absent in Caucasian populations.

In our study, the most common mutation associated with slow acetylation

(341T>C) occurs with a significantly higher frequency (0.364) than other mutant

variants and it was similar to what had been reported in other ethnic groups.

Moroccan, Egyptian, Spanish, Omani, Saudi Arabian, American and German

populations reported high frequencies of allele NA T2*5 between (42% - 53%),

whereas Argentine, African, Indian, Senegalese, Tunisian and Southern Brazil

populations reported (28% - 37%). However, it was at a very low frequency in Asian

populations « 7%) (Meyer U and Zanger U 1997, Guaoua S et aI 2014).

Indian population reported the highest frequency of allele NA T2 *6 (38%)

whereas, German, Omani, American, Egyptian, Argentina, Moroccan, Spanish, 129 Japanese and Southern Korean populations have frequencies ranging between 22% - 28.5%. However, in Senegalese, Tunisian, African and Southern Brazil populations it was at a very low frequency (10% - 18%) (Meyer U and Zanger U 1997, Guaoua S et al 2014).

In regard to allele NA T2*7 the frequency was the highest in Tunisian and

Southern Korean populations 15% and 13.2%, respectively. However, it markedly declined with Argentine, African, Omani, Indian, Egyptian, Southern Brazil and

Moroccan populations to (2% - 8%), although it was found to be negligible in

American, German, Japanese, Spanish and Senegalese populations «1%) (Meyer

U and Zanger U 1997, Guaoua S et aI 20 14).

African, Senegalese, Tunisian and Moroccan populations reported the

highest frequency of allele NA T2 *14 between (4% - 10.3%) where it was found to be at low level with other populations « 2%) (Meyer U and Zanger U 1997, Guaoua S et al 2014).

Moreover, in one of our previous studies conducted by Wool house et al

(1997) we had determined the polymorphic N-acetyltransferase (NA T2) genotypes

in 106 unrelated Emiratis from Eastern region of Abu Dhabi by PCR-RFLP analysis

(Wool house et al 1997). We found more than 13 different genotypes, 4 associated

with the rapid acetylator phenotype and 9 with the slow acetylator phenotype. The

prevalence of slow acetylation alleles were 81.6%. In our study, we reported 27

different genotypes, 9 associated with the rapid acetylator phenotype and 18 with

the slow acetylator phenotypes and the preva lence of slow acetylation alleles were

86.6%. The most common allele was NA T2*5.

Interestingly, we were able to report the presence of 3 new variants

"510T>C, 775A>C and 733A>C" that have been confirmed by DNA sequencing and

were associated with slow acetylation activity. These nucleotide changes represent 130 new allele NA T2*5 subtype and new allele NA T2*6 subtype as following: NA T2*5 new subtype: 282C>T, 341T>C, 481C>T, 590G>A, 803G>A, 775A>C and 733A>C variants and NA T2*6 sUbtype: contains 282C> T, 775A>C, 590G>A, 510T>C and

803G>A variants.

Hence, when Hardy-Weinberg equation was applied, the apparent numbers of homozygous and heterozygous slow acetylators were not in strict accordance with the Hardy-Weinberg equilibrium for allele NA T2*7 and NA T2 *14, which is a clear indication that individual genotypes cannot be predicted, although, for allele

NA T2*5 and NA T2*6 the result was consistent with Hardy-Weinberg Equilibrium.

This finding may be due to the evolutionary influences, such as mate choice, mutation, selection, genetic drift, gene flow and meiotic drive, since one or more of these influences are typically present in real populations. The level of consanguinity in the UAE is very high where more than 50% of marriages are consanguineous.

Indeed, about 50% of the sample group reported consanguinity of their parents. This clearly influences the genotypes frequencies and their deviation from Hardy­

Weinberg.

Our samples have represented few numbers of those with PMH and drug sensitivity and when they have been tested for their relation and effect on genotype we found that the results were not supported by statistical evidence. Nevertheless, we also found the results were not statistically significant and no differences have been found between cities in regard to genotype and alleles distribution between the different Emirates. 131

Chapter 6: Conclusions

6.1 CYP1A2

The frequency of slow activity CYP1A2 enzyme alleles is very low among

Emiratis which correlates with the presence of low frequencies of mutant alleles in

CYP1A2 gene. The genotype frequency of the wild type allele is the highest in this population, followed by CYP1 A2*1 Al"1C and CYP1A2*1 A/*1 K genotypes, respectively.

Those who are homozygote for alleles CYP1A2*1C and CYP1A2*3 might be at high risk of toxicity with some drugs and some diseases as these alleles are associated with the slowest phenotype status. Consequently, genetic testing is recommended prior to prescribing medications that are largely metabolized by

CYP1A2.

6.2 NA T2

There is a high percentage of slow acetylators among Emiratis which correlates with the presence of high frequencies of mutant alleles in NA T2 gene.

The genotype frequency of the allele NA T2 *5 was the highest in this population.

Moreover, the genotype frequency of NA T2*58/*78, NA T2 *58/*6A, NA T2 *781* 148 and NA T2*41*58 were the highest in this population.

I ndividuals who carried NAT2*6AJ*6A, NAT2*6AJ*7B, NAT2*7B/*7B,

NAT2*5AJ*5B or NAT2*5AJ*5A genotypes might be at higher risk of toxicity with drugs and some diseases compared to others as these genotypes are associated with the slowest acetylation status. Consequently, genetic testing is recommended prior to prescribing medications that are largely metabolized by NAT2. 132

References

Abe M, Suzuki T, Deguchi T (1993). An improved method for genotyping of N­ acetyltransferase polymorphism by polymerase chain reaction. Jpn. J. Hum. Genet. 38: 163-168.

Afonina I, ZivartsM , Kutyavin I, ( ...), Meyer RB (1997). Efficient priming of PCR with shortoli gonucleotides conjugated to a minor groove binder. Nucleic Acids Res. 25:2657-2660.

Agundez JA, Golka K, Martinez C, ( ...), Garda-Martin E (2008). Unravelling ambiguous NAT2 genotyping data. Clin Chem 54: 1 390-1 394.

Agundez JAG, Martinez C, Olivera M, ( ... ), Benitez J (1998). Expression in human prostate of drug-and carcinogen-metabolizing enzymes: association with prostate cancer risk. Br. J. Cancer, 78: 1361-1 367.

Aitchison KJ, Gonzalez FJ, ( ...), Quattrochi LC (2000). Identification of novel polymorphisms in the 5' flanking region of CYP1A2, characterization of interethnic variability, and investigation of their functional significance. Pharmacogenetics. 10: 695-704.

Akagah B, Lormier AT, Fournet A, Figadere B (2008). Oxidation of antiparasitic 2- substituted quinolines using metalloporphyrin catalysts: scale-up of a biomimetic reaction for metabolite production of drug candidates. Org. Biomol. Chem. 6 (24): 4494-4497.

Aklillu E, Carrillo JA, Makonnen E, ( ... ), Ingelman-Sundberg M (2003). Genetic polymorphism of CYP1 A2 in Ethiopians affecting induction and expression: characterization of novel haplotypes with single-nucleotide polymorph isms in intron 1. Mol Pharmacol. 64(3): 659-669.

Aliorge 0, Chevalier 0, Lo-Guidice JM, ( ...), Broly F (2003). Identification of a novel splice-site mutation in the CYP1 A2 gene. Br J Clin Pharmacol. 56(3): 341 -344.

Altayli E, Gunes S, Yilmaz AF, Goktas S, Bek Y (2009). CYP1A2, CYP2D6, GSTM1, GSTP1, and GSTT 1 gene polymorphisms in patients with bladder cancer in a Turkish population. Int Urol Nephrol. 41: 259-266.

Ambrosone CB, Freudenheim JL, Graham S, ( ... ), Shields PG (1996). Cigarette smoking, N-acetyltransferase 2 genetic polymorphisms, and breast cancer risk. J. Am. Med. Assoc. , 276: 1494-1 501.

Ambrosone CB, Freudenheim JL, Sinha R, ( ...), Shields PG (1998). Breast cancer risk, meat consumption and N-acetyltransferase (NA T2) genetic polymorphisms. Int. J. Cancer, 75: 825-830.

An HR, Wu XQ, Wang ZY, Zhang JX, Liang Y (2012). NA T2 and CYP2E1 polymorphisms associated with antituberculosis drug-induced hepatotoxicity in 133 Chinese patients. Clin Exp Pharmacol Physio/' 39(6): 535-543.

Bandmann 0, Vaughan JR, Holmans P, Marsden CD, Wood NW (2000). Detailed genotyping demonstrates association between the slow acetylator genotype for N­ acetyltransferase 2 (NA T2) and familial Parkinson's disease. Mov Disord. 15: 30- 35

Batra J and Ghosh B (2008). N-acetyltransferases as markers for asthma and allergic/atopic disorders. Curr Drug Metab. 9(6): 546-553.

Bebia Z, Buch SC, Wilson JW, ( ... ), Branch RA (2004). Bioequivalence revisited: influence of age and sex on CYP enzymes. Clin. Pharmacol. Th er.76(6): 618-627.

Begas E, Kouvaras E, Tsakalof A, Papakosta S, Asprodini EK (2007). In vivo evaluation of CYP1A2, CYP2A6, NAT-2 and xanthine oxidase activities in a Greek population sample by the RP-HPLC monitoring of caffeine metabolic ratios. Biomed Chromatogr. 21 (2): 190-200.

Bell DA, Stephens E, Castranio T, ( . ..) , Strange RC (1995). Polyadenylationpolymorphism in the N-acetyltransferase gene 1 (NAT1) increases risk of colorectal cancer. Cancer Res. , 55: 3537-3542.

Benowitz NL, Jacob P, Mayan H, Denaro C (1995). Sympathomimetic effects of paraxanthine and caffeine in humans. Clinical pharmacology and therapeutics. 58(6): 684-691.

Besarati Nia A, Van Straaten HW, Kleinjans JC, Van Schooten FJ (2000). Immunoperoxidase detection of 4-aminobiphenyl and polycyclic aromatic hydrocarbons DNA adducts in induced sputum of smokers and non-smokers. Mutat Res. 468: 125-135

Bilgen T, Tosun 0, Luleci G, Keser I (2008). Frequencies of four genetic polymorph isms in the CYP1A2 gene in Turkish population. Genetika. 44: 1133- 1136.

Bliden KP, Dichiara J, Lawai L, ( ... ), Baker BA (2008). The association of cigarette smoking with enhanced platelet inhibition by clopidogrel. J Am Coli Cardiol. 52: 531-533

. lopment of Bluhm RE, Adedoyin A, McCarver DG, Branch RA. (1999) Deve dapsone toxicity in patients with inflammatory dermatoses: activity of acetylation and hydroxylation of dapsone as risk factors. Clin Pharmacol Therapeut., 65: 598- 605

Blume M, Grant OM, McBride W, Heim M, Meyer UA (1990). Human arylamine N­ acetyltransferase genes: isolation, chromosomal localization, and functional expression. DNA Cell BioI. , 9: 193-203.

Bohanec GP, Rozman B, Tomsic M, ( ...) , Dolzan V (2008). Genetic polymorphism of CYP1A2 and the toxicity of leflunomide treatment in rheumatoid arthritis patients. 134 Eur J Clin Pharmacol. 64: 871-876.

Bolt HM, Selinski S, Dannappel 0, Blaszkewicz M, Golka K (2005). Re­ Investigation of the concordance of human NA T2 phenotypes and genotypes. Arch Toxicol. 79(4): 196-200.

Bose PO, Sarma MP, Medhi S, ( ... ), Kar P (201 1). Role of polymorphic N-acetyl transferase2 and cytochrome P4502E 1 gene in antituberculosis treatment-induced hepatitis. J Gastroenterol Hepatol. 26(2): 312-320.

Bouchardy C, Mitrunen K, Wikman H, ( ...), Hirvonen A (1998). N-Acetyltransferase NA T1 and NAT2 genotypes and lung cancer risk. Pharmacogenetics, 8: 291-298.

Boukouvala S and Fakis G (2005). Arylamine N-acetyltransferases: what we learn from genes and genomes. Drug Metab Rev. 37(3): 51 1 -564.

Boukouvala S and Sim E (2005). Structural analysis of the genes for human arylamine N-acetyltransferases and characterisation of alternative transcripts. Basic Clin Pharmacol Toxicol. 96(5): 343-351.

Brockton N, little J, Sharp L, Cotton SC (2000). N-acetyltransferase polymor­ phisms and colorectal cancer: a HuGE review. Am J Ep idemiol. 151: 846-861

Brockmoller J, Cascorbi I, Kerb R, Roots I (1996). Combined analysis of inherited polymorphisms in arylamine N-acetyltransferase 2, glutathione-S-transferases M1 and T1 , microsomal epoxide hydrolase, and cytochrome P450 enzymes as modulators of bladder cancer risk. Cancer Res., 56: 3915-3925.

Browning SL, Tarekegn A, Bekele E, Bradman N, Thomas MG (2010). CYP1A2 is more variable than previously thought: a genomic biography of the gene behind the human drug-metabolizing enzyme. Pharmacogenet. Genomics 20: 647-664.

Buck ML (1997). The cytochrome P450 enzyme system and its effect on drug metabolism. Pediatric Pharmacotherapy. 3(5).

Bulovskaya LN, Krupkin RG, Bochina TA, Shipkova AA, and Pavlova MV (1978). Acetylator phenotype in patients with breast cancer. Oncology (Basel), 35: 185- 188.

Burgess EJ, and Trafford JA (1985). Acetylator phenotype in patients with lung carcinoma: a negative report. Eur. J. Respir. Dis. , 67: 17-19.

Butcher NJ, Boukouvala S, Sim E and Minchin R F ( 2002). Pharmacogenetics of the arylamine N-acetyltransferases. Pharmacogenomics J. 2(1 ):30-42.

Butcher NJ and Minchin RF (2012). Arylamine N-acetyltransferase 1: a novel drug target in cancer development. Pharmacological re views. 64( 1): 147-165

Butler MA, Lang NP, Young JF, ( ...), Kadlubar FF (1992). Determination of CYP1A2 and NAT2 phenotypes in human populations by analysis of caffeine 135 urinary metabolites. Pharmacogenetics; 2(3): 116-127.

Carrillo JA, Christensen, ( ... ), Leif (2000). Evaluation of Caffeine as an In Vivo Probe for CYP1A2 Using Measurements in Plasma, Saliva, and Urine. Th erapeutic Drug Monitoring; 22 (4): 409-417

Cartwright RA, Glashan RW, Rogers HJ, C ... ), Kahn M (1982). A role of N­ acetyltransferase phenotypes in bladder carcinogenesis: a pharmacogenetic epidemiological approach to bladder cancer. Lancet, 2: 842-846.

Cascorbi I, Brockmoller J, Mrozikiewicz PM, Muller A, Roots I (1999). Arylamine N­ acetyltransferase activity in man. Drug Metab Rev. 31 (2): 489-502.

Cascorbi I, Drakoulis N, Brockmoller J, ( ... ), Roots 1 (1995). Arylamine N­ acetyltransferase (NAT2) mutations and their allelic linkage in unrelated Caucasian individuals: correlation with phenotypic activity. Am J Hum Genet. 57(3): 581-592.

Cascorbi I ( 2006). Genetic basis of toxic reactions to drugs and chemicals. Toxico/ Lett; 162: 16-28.

Cascorbi I and Roots I (1999). Pitfalls in N-acetyltransferase 2 genotyping. Pharmacogenetics, 9: 123-1 27.

Cascorbi I, Brockmoller J, Mrozikiewicz PM, ( ... ), Roots I (1996). Homozygous rapid arylamine N-acetyltransferase (NAT2) genotype as a susceptibility factor for lung cancer. Cancer Res., 56: 3961 -3966.

Chamorro JG, Castagnino JP, Musella RM, ( ... ), de LarrafiagaGF. (2013). Sex, ethnicity, and slow acetylator profile are the major causes of hepatotoxicity induced by antituberculosis drugs. J Gastroenterol Hepatol. 28(2): 323-328.

Chan DKY, Lam MKP, Wong R, Hung WT,Wilcken DEL (2003). Strong association between N-acetyltransferase 2 genotype and PO in Hong Kong Chinese. Neurology 60: 1002-1005.

Chen J, Stampfer MJ, Hough HL, ( ...), Hunter OJ (1998). A prospective study of N­ acetyltransferase genotype, red meat intake, and risk of colorectal cancer. Cancer Res. , 58: 3307-331 1.

Chevalier 0, Cauffiez C, Allorge 0, ( . ..), Broly F (2001). Five novel natural allelic variants-951A>C, 1 042G>A (D348N), 1156A>T (1386F), 121 7G>A (C406Y) and 1291 C>T (C431Y)-of the human CYP1A2 gene in a French Caucasian population. Hum Mutat. 17(4): 355-356.

Chida M, Yokoi T, Fukui T, ( ... ), Kamataki T (1999). Detection of three genetic polymorphisms in the 5'-flanking region and intron 1 of human CYP1A2 in the Japanese population. Jpn J Cancer Res. 90: 899-902

Choudhary C, Kumar C, Gnad F, ( ... ), Mann M (2009). Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions. Science 325 136 (5942): 834-840.

Christiansen L, Bygum A, Jensen A, ( ...), Petersen NE (2000). Association between CYP1A2 polymorphism and susceptibility to porphyria cutanea tarda. Hum. Genet. 107: 612-614.

Chung CC, Magalhaes WCS, Gonzalez-Bosquet J, and Chanock SJ (2010). Genome-wide association studies in cancer-current and future directions. Carcinogenesis. 31 (1): 111-120.

Clark OW (1985). Genetically determined variability in acetylation and oxidation. Therapeutic implications Drugs. 29: 342-375

Corchero J, Pimprale S, Kimura S, Gonzalez FJ (2001). Organization of the CYP1A cluster on human chromosome 15: implications for gene regulation. Pharmacogenetics. 11: 1-6.

Cornelis MC, EI-Sohemy A, Kabagambe EK, Campos H (2006). Coffee, CYP1A2 genotype, and risk of myocardial infarction. JA MA . 295:11 35-1 141.

Cornel is MC, Monda KL, Yu K, ( ... ), Bennett SN. (201 1). Genome-wide meta­ analysis identifies regions on 7p21 (AHR) and 15q24 (CYP1A2) as determinants of habitual caffeine consumption. PLoS Genet. 7 (4):e1 002033.

Cribb AE, Nuss CE, Alberts OW, ( . .. ), Grossman SJ. (1996). Covalent binding of sulfamethoxazole reactive metabolites to human and rat liver subcellular fractions assessed by immunochemical detection. Chem Res Toxico/ 9: 500-507

Dai Z, Papp AC, Wang 0, Hampel H, Sadee W (2008). Genotyping panel for assessing response to cancer chemotherapy. BMC Medical Genomics 1: 24.

Daly AK and Day CP (2012). Genetic association studies in drug-induced liver injury. Drug Metab Rev. 44(1): 116-126.

Das KM and Dubin R (1976). Clinical pharmacokinetics of sulphasalazine. Clin Pharmacokinet. 1: 406-425.

Davis CM and Shearer WT (2008). Diagnosis and management of HIV drug hypersensitivity. J Allergy Clin Immunol. 121 (4): 826-832.

Degtyarenko KN (1995). Structural domains of P450-containing systems. Protein Engineering. 8(8): 737-747.

Deitz AC, Zheng W, Leff MA, ( . ..), Hein OW (1999). N-Acetyltransferase-2 (NAT2) acetylation polymorphism, well-done meat intake and breast cancer risk among post-menopausal women. Proc. Am. Assoc. Cancer Res., 40: 148.

Delomenie C, Fouix S, Longuemaux S, ( ...), Picard B (2001). Identification and functional characterisation of arylamine N-acetyltransferase in eubacteria: evidence for highly selective acetylation of 5-aminosalicylic acid. J Bacteriol. 183: 341 7- 137 3427.

Djordjevic N, Carrillo JA, Roh HK, ( ... ), Aklillu E (2012). Comparison of N­ acetyltransferase-2 enzyme genotype-phenotype and xanthine oxidase enzyme activity between Swedes and Koreans. J Clin Pharmacol. 52(10): 1527-1 534.

Djordjevic N, Carrillo JA, Ueda N, ( ... ), Aklillu E (201 1). N-Acetyltransferase-2 (NA T2) gene polymorphisms and enzyme activity in Serbs: unprecedented high prevalence of rapid acetylators in a White population. J Clin Pharmacol. 51 (7): 994- 1003.

Djordjevic N, Ghotbi R, Bertilsson L, Jankovic S, Aklillu E (2008). Induction of CYP1A2 by heavy coffee consumption in Serbs and Swedes. Eur J Clin Pharmacal. 64: 381 -385

Djordjevic N, Ghotbi R, Jankovic S, Aklillu E (2010). Induction of CYP1A2 by heavy coffee consumption is associated with the CYP1A2 - 163C > A polymorphism. Eur J Clin Pharmacal. 66: 697-703.

Dossing M., Pilsgaard H., Rasmussen B. & Poulsen H.E. (1983). "Time course of phenobarbital and cimetidine mediated changes in hepatic drug metabolism." Eur J Clin Pharmacal. 25:215-22.

Dresser GK, Spence JD, Bailey DG (2000). Pharmacokinetic-pharmacodynamic consequences and clinical relevance of cytochrome P450 3A4 inhibition. Clin Pharmacakinet, 38:41 -57.

Dr6zdz M, Gierek T, Jesdryczko A, Pilch J and Piekarska J (1987). Nacetyltransferase phenotype of patients with cancer of the larynx. Neaplasma (Bratisl.), 34: 481-484.

Eap CB, Bender S, Jaquenoud Sirot E, ( ...), Baumann P (2004). Nonresponse to clozapine and ultrarapid CYP1A2 activity: clinical data and analysis of CYP1A2 gene. J Clin Psychapharmacal. 24:214-219.

Ebisawa T and Deguchi T (1991). Structure and restriction fragment length polymorphism of genes for human liver arylamine N-acetyltransferases. Biachem Biaphys Res Cammun. 177(3):1 252-1257.

Eichelbaum M, Ingelman-Sundberg M, Evans WE (2006). Pharmacogenomics and individualized drug therapy. Annu Rev Med. 57:119-137.

Evans DAP (1984). Survey of the human acetylator polymorphism in spontaneous disorders. J Med Genet. 21: 243-253

Evans DAP (1993). Genetic Factors in Drug Therapy: Clinical and Molecular Pharmacogenetics. Cambridge, UK: Cambridge University. Page 21 1-285.

Fernandes MR, de Carvalho DC, dos Santos AK, ( ... ), dos Santos NP (2013). Association of slow acetylation profile of NAT2 with breast and gastric cancer risk 138 In Brazil Anticancer Res. 33(9): 3683-3689.

Figueroa JD, Ye Y, Siddiq A, ( oo.) . Rothman N (2014). Genome-wide association study identifies multiple loci associated with bladder cancer risk. Hum Mol Genet. 1;23(5): 1387-1 398.

Filiadis IF, Georgiou I, Alamanos Y, ( oo.), lolis 0 (1999). Genotypes of N­ acetyltransferase-2 and risk of bladder cancer: a case-control study. J. Ural. ; 161: 1672-1675.

Fontana RJ, Turgeon OK, Woolf TF, ( oo.), Watkins PB (1996). The caffeine breath test does not identify patients susceptible to tacrine hepatotoxicity. Hepato/ogy. 23(6): 1 429-35.

Freeman Cl, Swords R, Giles FJ (2012). Amonafide: a future in treatment of resistant and secondary acute myeloid leukemia? Expert Rev Hematol. 5(1): 17-26.

Fritz KS, Galligan JJ, Hirschey MD, Verdin E, Petersen DR (2012). Mitochondrial Acetylome Analysis in a Mouse Model of Alcohol-Induced Liver Injury Utilizing SI RT3 Knockout Mice. Journalof Proteome Research 11 (3): 1633-1 643.

Fukino K, Sasaki Y, Hirai S, ( oo.), Ueno K (2008).Effects of N-acetyltransferase 2 (NAT2), CYP2E1 and Glutathione-S-transferase (GST) genotypes on the serum concentrations of isoniazid and metabolites in tuberculosis patients. J Toxicol Sci. 33(2): 187-195.

Fuselli S, Gilman RH, Chanock SJ, ( oo.), Tarazona-Santos E (2007). Analysis of nucleotide diversity of NA T2 coding region reveals homogeneity across Native American populations and high intra-population diversity. Pharmacogenomics J. 7(2): 144-1 52.

Gertig OM, Hankinson SE, Hough H, ( oo.), Hunter OJ (1999). N-Acetyltransferase 2 genotypes, meat intake and breast cancer risk. Int. J. Cancer, 80: 13-1 7.

Ghotbi R, Christensen M, Roh HK, ( ... ), Bertilsson l (2007). Comparisons of CYP1A2 genetic polymorphisms, enzyme activity and the genotype-phenotype relationship in Swedes and Koreans. Eur J Clin Pharmacol. 63: 537-546.

Gil JP and Lechner MC (1998). Increased frequency of wild-type arylamine- N­ acetyltransferase allele NAT2*4 homozygotes in Portuguese patients with colorectal cancer. Carcinogenesis (lond.), 19: 37-41.

oo. E (1998). Genetic polymorphism of Gonzalez MV, Alvarez V, Pello MF, ( ), Cota N-acetyltransferase-2, glutathione Stransferase- M1, and cytochromes P450llE1 and P450llB in the susceptibility to head and neck cancer. J. Clin. Pathol. , 51: 294- 298.

Gonzalez-Fierro A, Vasquez-Bahena 0, Taja-Chayeb l, ( oo.) , Duenas-Gonzalez A (2011). Pharmacokinetics of hydralazine, an antihypertensive and DNA­ demethylating agent, using controlled-release formulations designed for use in dosing schedules based on the acetylator phenotype. Int J Clin Pharmacol Ther. 139 49(8): 519-524.

Grant OM. Blume M. Beer M. Meyer UA (1991). Monomorphic and polymorphic human arylamine N-acetyltransferases: a comparison of liver isozymes and expressed products of two cloned genes. Mol Pharmacol, 39: 184-1 91

Grant OM. Hughes NC. Janezic SA. ( ...). Grewal R (1997). Human acetyltransferase polymorphisms. Mutat. Res .• 376: 61-70.

Grant OM. Mbrike K. Eichelbaum M and Meyer UA (1990). Acetylation pharmacogenetics. The slow acetylator phenotype is caused by decreased or absent arylamine N-acetyltransferase in human liver. J Clin Invest., 85(3): 968-972.

Grant OM. Tang BK. Kalow W (1984). A simple test for acetylator phenotype using caffeine. Br J C!in Pharmacol. 17(4): 459-464.

Gremmel T. Steiner S. Seidinger D. ( ... ). Kopp CW (2009). Smoking promotes clopidogrel-mediated platelet inhibition in patients receiving dual antiplatelet therapy. Thromb Res. 124: 588-591.

Guaoua S. Ratbi I. Laarabi FZ. ( ...) Sefiani A (2014). Distribution of allelic and genotypic frequencies of NA T2 and CYP2E 1 variants in Moroccan population. BMC Genetics. 15: 156.

Guengerich FP. Parikh A. Turesky RJ, Josephy PO (1999). Inter-individual differences in the metabolism of environmental toxicants: cytochrome P450 1A2 as a prototype. Mutat Res. 428(1-2): 115-124

Guengerich FP (2001). Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. Chem Res Toxico!. 14:61 1-650.

Guengerich FP (2006). Cytochrome P450s and other enzymes in drug metabolism and toxicity. AAPS J, 8: E101-E1 11.

Guessous I, Oobrinas M. Kutalik Z, ( . . . ). Murielle Bochud (2012). Caffeine intake and CYP1A2 variants associated with high caffeine intake protect non-smokers from hypertension. Human Molecular Genetics; 21 (14): 3283-3292.

Gunes A and Dahl ML (2008). Variation in CYP1A2 activity and its clinical implications: influence of environmental factors and genetic polymorphisms. Pharmacogenomics. 9: 625-637.

Gupta VH. Amarapurkar ON. Singh M. ( ...) . Wangikar PP (2013). Association of N­ acetyltransferase 2 and cytochrome P450 2E1 gene polymorphisms with antituberculosis drug-induced hepatotoxicity in Western India. J Gastroenterol Hepatol. 28(8): 1368-1 374.

ur Hamdy SI. Hiratsuka M. Narahara K, ( . ..). Moursi N (2003). Genotyping of fo Br J Clin genetic polymorphisms in the CYP1A2 gene in the Egyptian population. Pharmacol. 55: 321 -324 140

Hartter S. Korhonen T. ( ... ). Lundgren S (2006). Effect of caffeine intake 12 or 24 hours prior to melatonin intake and CYP1A2*1 F polymorphism on CYP1A2 phenotyping by melatonin. Basic Clin Pharmacol Toxicol. 99: 300-304.

He X. Wei J. Lui Z. Xie J. Wang W, Ou Y, Chen Y, Si H. Liu Q, Xia L and Wei W (2014). Association between CYP1A2 and CYP1 B1 Polymorphisms and Colorectal Cancer Risk: A Meta-Analysis. PL OS ONE 9(8): e100487.

Hedrick P (2011). Genetics of Populations. Jones & Bartlett Learning. pp. 104. ISBN 978-0-7637-5737 -3.

Hein OW (1988). Acetylator genotype and arylamine-induced carcinogenesis. Biochim. Biophys. Acta, 948: 37-66.

Hein OW (2009). N-acetyltransferase SNPs: emerging concepts serve as a paradigm for understanding complexities of personalized medicine. Expert Opin Drug Metab Toxicol. 5(4): 353-366.

Hein OW, Doll MA. Fretland AJ, ( ... ), Xiao GH (2000). Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorph isms. Cancer

Epidemio/, Biomarkers & Prevention, 9:29-42

Hein OW, Doll MA, Rustan TO, Ferguson RJ (1995). Metabolic activation of N­ hydroxyarylamines and N-hydroxyarylamides by 16 recombinant human NAT2 allozymes: effects of 7 specific NAT2 nucleic acid substitutions. Cancer Res. 15;55(16): 3531-3536.

Hein OW, Ferguson RJ, Doll MA, Rustan TO, and Gray K (1994). Molecular genetics of human polymorphic N-acetyltransferase: enzymatic analysis of 15 recombinant human wild-type, mutant, and chimeric NAT2 allozymes. Hum. Mo/. Genet. , 3: 729-734.

Hein OW, Grant OM, and Sim E (2000). Update on consensus arylamine N­ acetyltransferase gene nomenclature. Pharmacogenetics, 10 (4): 291-292

Hein OW, Boukouvala S, Grant OM, Minchin RF and Sim E (2008). Changes in consensus arylamine N-acetyltransferase gene nomenclature. Pharmacogenet. Genomics, 18(4): 367-368.

Henning S, Cascorbi I, Munchow B, Jahnke V and Roots I (1999). Association of arylamine N-acetyltransferases NAT1 and NAT2 genotypes to laryngeal cancer risk. Pharmacogenetics, 9: 103-1 11.

Hickman 0, Risch A, Buckle V, ( ...), Sim E (1994). Chromosomal localization of human genes for arylamine N-acetyltransferase. Biochem J. 1; 297 (Pt 3):441-445.

(1991). N-acetyltransferase polymorphism: Comparison of Hickman 0 and Sim E Issue phenotype and genotype in humans. Biochemical Pharmacology. Volume 42, 5, Pages 1007-1014. ited GSTM1 Hirvonen A. Pelin K, Tammilehto L, ( ...), Linnainmaa K (1995) . Inher 141 and NAT2 defects as concurrent risk modifiers in asbestos-related human malignant mesothelioma. Cancer Res., 55: 2981-2983.

Hirvonen A, Saarikoski ST, Linnainmaa K, ( ...), Vainio H (1996a). Glutathione S­ transferase and Nacetyltransferase genotypes and asbestos-associated pulmonary disorders. J. Natl. Cancer Inst. , 88: 1853-1 856.

Hirvonen A, Saarikoski ST, Linnainmaa K, ( ... ), Vainio HJ (1996b). Lutathione S­ transferase and Nacetyltransferase genotypes and asbestos associated pulmonary disorders. J Nat!. Cancer Insf.; 88 (24): 1853-1856.

Ho HT, Wang TH, Hsiong CH, ( ...), Hu OY (2013). The NAT2 tag SNP rs1 495741 correlates with the susceptibility of antituberculosis drug-induced hepatotoxicity. Pharmacogenet Genomics. 23(4): 200-207.

Homolya L, Varadi A, Sarkadi B (2003). Multidrug resistance-associated proteins: Export pumps for conjugates with glutathione, glucuronate or sulfate. BioFactors 1 7 (1-4): 1 03-1 14. https://galaxc.sengenics.com/login;jsessionid=73C4635A65F960E1 00B40CB1 037 39BA8 http://www. . orgl http://www.genenames.org/ http://www.imm.ki .se/CYPalieles http://www. ncbi. nlm . nih.gov/ge ne/1 544 http://www.snpedia.com/index.php/CYP1A2

Huang JO, Guo WC, Lai MO, Guo YL, Lambert GH (1999). Detection of a novel cytochrome P-450 1 A2 polymorphism (F21 L) in Chinese. Drug Metab Dispos. 27(1): 98-101.

Hubbard AL, Harrison OJ, Moyes C, ( ...), Smith CAD (1997). N-Acetyltransferase 2 genotype in colorectal cancer and selective gene retention in cancers with chromosome 8p deletions. Gut, 41: 229-234.

Hughes HB, Biehl JP, Jones AP and Schmidt LH (1954). Metabolism of isoniazid in man as related to the occurrence of peripheral neuritis. Am. Rev. Res. Dis. , 70: 266-273.

Hunter OJ, Hankinson SE, Hough H, ( . . . ), Kelsey K (1997). A prospective study of NAT2 acetylation genotype, cigarette smoking and risk of breast cancer. Carcinogenesis (Lond.), 18: 2127-2132.

Husain A, Barker OF, States JC, Doll MA, Hein OW (2004). Identification of the major promoter and non-coding exons of the human arylamine N-acetyltransferase 1 gene (NAT1). Pharmacogenetics. 14(7): 397-406. 142

Husain A, Zhang X, Doll MA, ( ...), Hein OW (2007). Identification of N­ acetyltransferase 2 (NAT2) transcription start sites and quantitation of NAT2- specific mRNA in human tissues. Drug Metab Dispos. 35(5): 721-727.

Ikeya K, Jaiswal AK, Owens RA, ( ... ), Kimura S (1989). Human CYP1A2: sequence, gene structure, comparison with the mouse and rat orthologous gene, and differences in liver 1A2 mRNA expression. Mo/ec. Endo cr. 3: 1399-1408.

Ilett KF, David BM, Detchon P, Castleden WM, Kwa R (1987). Acetylation phenotype in colorectal carcinoma. Cancer Res. 47: 1466-1469.

Ingelman-Sundberg M, Sim SC, Gomez A, Rodriguez-Antona C (2007). Influence of cytochrome P450 polymorphisms on drug therapies: pharmacogenetic, pharmacoepigenetic and clinical aspects. Pharmacol Ther. 116: 496-526.

Ingelman-Sundberg M (2001 a). Genetic susceptibility to adverse effects of drugs and environmental toxicants. The role of the CYP family of enzymes. Mutat Res. 482: 11-19.

Ingelman-Sundberg M (2001 b). Pharmacogenetics: an opportunity for a safer and more efficient pharmacotherapy. J Intern Med; 250: 186-200.

Ingelman-Sundberg M (2002). Polymorphism of cytochrome P450 and xenobiotic toxicity. Toxicology; 181-182: 447-452

Innocenti F, Iyer L, Ratain MJ (2001). Pharmacogenetics of anticancer agents: lessons from amonafide and irinotecan. Drug Metab Dispos. 29(4 Pt 2): 596-600.

Israili ZH, Dayton PG (1977). Metabolism of hydralazine. Drug Metab Rev. 6(2): 283-305.

Jain M, Kumar S, Lal P, ( ...), Mittal B (2007). Association of genetic polymorphisms of N-acetyltransferase 2 and susceptibility to esophageal cancer in north Indian population. Cancer Invest. 25(5): 340-346.

Jetter A, Kinzig-Schippers M, IIlauer M, ( ...), Fuhr U (2004). Phenotyping of N­ acetyltransferase type 2 by caffeine from uncontrolled dietary exposure. Eur J Clin Pharmacol. ; 60(1): 17-21.

Jorge-Nebert LF, Jiang Z, Chakraborty R, ( ...), McGarvey ST (2010). Analysis of human CYP1A1 and CYP1A2 genes and their shared bidirectional promoter in eight world populations. Hum Mutat. 31: 27-40.

Joshua EM, Brian P, Wayne K, ( ... ), John PR (2008). Comparison of CYP1A2 and NAT2 Phenotypes between Black and White Smokers. Biochem Ph armacol. 76(7): 929-937.

Jourenkova-Mironova N, Wikman H, Brouchardy C,( ... ), Hirvonen A (1999). Role of arylamine N-acetyltransferase 1 and 2 (NAT1 and NAT2) genotypes in susceptibility to oral/pharyngeal and laryngeal cancers. Pharmacogenetics, 9: 533- 143 537

Kagaya H, Miura M, Niioka T, ( ... ), Satoh S (2012). Influence of NAT2 polymorphisms on sulfamethoxazole pharmacokinetics in renal transplant recipients. Antimicrob Agents Chemother. 56(2): 825-829.

Kall MA and Clausen J (1995). Dietary effect on mixed function P450 1A2 activity assayed by estimation of caffeine metabolism in man. Hum Exp Toxicol. 14: 801- 807.

Kalow W, Tang BK (1993). The use of caffeine for enzyme assays: a critical appraisal. Clinical pharmacology and therapeutics; 53 (5): 03-514.

Kalow W, Tang BK (1993). The use of caffeine for enzyme assays: a critical appraisal. Clin Pharmacol Ther. 53: 503-514.

Kampman E, Slattery ML, Bigler J, ( ...), Potter JD (1999). Meat consumption genetic susceptibility and colon cancer risk: a United States multicenter case­ control study. Cancer Epidemiol. BiomarkPrev. , 8: 15-24.

Kaufmann GR, Wenk M, Taeschner W, ( ...), Haefeli W (1996). N-acetyltransferase 2 polymorphism in patients infected with human immunodeficiency virus. Clin Pharmacol Ther. 60(1 ):62-67.

Khan N, Pande V, Das A (2013). NAT2 sequence polymorphisms and acetylation profiles in Indians. Pharmacogenomics. 14(3): 289-303.

Kirlin WG, Ogolla F, Andrews AF, ( ...), Hein OW (1991). Acetylator genotype­ dependent expression of arylamine N-acetyltransferase in human colon cytosol from noncancer and colorectal cancer patients. Cancer Res., 51: 549-555.

Klein K, Winter S, Turpeinen M, Schwab M, Zanger UM. (2010). Pathway-targeted pharmacogenomics of CYP1A2 in human liver. Front Pharmacol. 1 :129.

Krusekopf S, Roots I, Hildebrandt AG, Kleeberg U (2003).Time-dependent transcriptional induction of CYP1A1, CYP1A2 and CYP1 B1 mRNAs by H+/K+ - ATPase inhibitors and other xenobiotics. Xenobiotica. 33(2): 107-1 18.

Kuhn UD, Anschutz M, Schmucker K, ( ... ), Blume HH. (2010). Phenotyping with sulfasalazine - time dependence and relation to NAT2 pharmacogenetics. Int J Clin Pharmacol Ther. 48(1): 1-10.

Kusumoto M, Ueno K, Oda A, ( ... ), Tanaka K (2001). Effect of fluvoxamine on the pharmacokinetics of mexiletine in healthy Japanese men. Clin. Ph armacol. Ther. ; 69(3): 104-107.

Ladero JM (2008). Influence of polymorphic N-acetyltransferases on non-malignant spontaneous disorders and on response to drugs. Curr Orug Metab. 9(6): 532-537.

Ladero JM, Gonzalez JF, Benitez J, ( ... ), Diaz-Rubio M (1991). Acetylator 144 polymorphism in human colorectal carcinoma. Cancer Res. , 51: 2098-2 100.

Lakowicz JR (1983). Principles of Fluorescence Spectroscopy. Plenum Press, New York, Page 303-339.

Lang NP and Kadlubar FF (1991). Aromatic and hetrocyclic amine metabolism and phenotyping in humans. Prog Clin BioI Res. 372: 33-47.

Lang NP, Chu DZJ, Hunter CF, ( ... ), Kadlubar FF (1986). Role of aromatic amine acetyltransferase in human colorectal cancer. Arch. Surg. 121: 1259-1261.

Layton OW, Bogen KT, Knize MG, ( ... ), Felton JS (1995). Cancer risk of heterocyclic amines in cooked foods: an analysis and implications for research. Carcinogenesis (Lond.), 16: 39-52.

Le Marchand L, Sivaraman L, Franke AA, ( ...), Cooney RV (1996). Predictors of N­ acetyltransferase activity: should caffeine phenotyping and NAT2 genotyping be used interchangeably in epidemiological studies? Cancer Epidemiol Biomarkers Prevo 5(6): 449-455.

Le Marchand L, Franke AA,Cus ter L, Wilkens LR, Cooney RV (1997). Lifestyle and nutritional correlates of cytochrome CYP 1 A2 activity: inverse associations with plasma lutein and alphatocopherol. Pharmacogenetics; 7: 11-19

Lee EJD, Zhao B, Seow-Choen F (1998). Relationship between polymorphism of N-acetyltransferase gene and susceptibility to colorectal carcinoma in a Chinese population. Pharmacogenetics 8: 513-517.

Leff MA, Fretland AJ, Doll MA and Hein OW (1999). Novel human N­ acetyltransferase 2 alleles that differ in mechanism for slow acetylator phenotype. J. BioI. Chem., 274: 34519-34522.

Leyland-Jones B, Banerjee K, Lang N and Wong P (1999). A reliable method for NAT2 phenotyping by ELISA. Proc. Am. Assoc. Cancer Res. , 40: 53.

Li N and Stephens M (2003). Modelling linkage disequilibrium and identifying recombination hotspot using single nucleotide polymorphism data. Genetics; 165: 2213-33. Erratum: Genetics. 167:1039.

Lin HJ, Han CY, Lin BK, Hardy S (1994). Ethnic distribution of slow acetylator mutations in the polymorphic N-acetyltransferase (NAT2) gene. Pharmacogenetics 4: 125-1 34.

Liston HL, Markowitz JS, DeVane CL (2001). "Drug glucuronidation in clinical psychopharmacology". J Clin Psychopharmacol .21 (5): 500-1 5.

Lorenzo B, and Reidenberg MM (1989). Potential artifacts in the use ofcaf fe ine to determine acetylation phenotype. Br. J. Clin. Pharmacol. , 28: 207-208.

Lubin JH, Kogevinas M, Silverman 0, ( ... ), Rothman N (2007). Evidence for an intensity dependent interaction of NAT2 acetylation genotype and cigarette 145 smoking in the Spanish Bladder Cancer Study. Int. J. Epidemiol. ; 36(1): 236-241.

Luca F, Bubba G, Basile M, ( . . . ), Novelletto A (2008). Multiple advantageous amino acid variants in the NAT2 gene in human populations. PLoS One. 5; 3(9): e31 36.

Ma JJ, Liu CG, Li JH, Cao XM, Sun SL, Yao X (2009). Effects of NAT2 polymorphism on SASP pharmacokinetics in Chinese population. Clin Chim Acta. ; 407(1-2): 30-35.

Magalon H, Patin E, Austerlitz F, ( ... ), Heyer E (2008). Population genetic diversity of the NAT2 gene supports a role of acetylation in human adaptation to farming in Central Asia. Eur J Hum Genet. 16(2): 243-2451.

Mahapatra S, Woolhiser LK, Lenaerts AJ, ( ...), Belisle JT (2012). A novel metabolite of antituberculosis therapy demonstrates host activation of isoniazid and formation of the isoniazid-NAD+ adduct. Antimicrob Agents Chemother. 56(1): 28- 35.

Malik MA, Upadhyay R, Modi DR, Zargar SA, Mittal B (2009). Association of NAT2 gene polymorph isms with susceptibility to esophageal and gastric cancers in the Kashmir Valley. Arch Med Res. 40(5):416-423.

Marini DM, Hastings SB, Brooks LR, ( ...), Kohlmeiter L (1997). Pilot study of free and conjugated urinary mutagenicity during consumption of pan-fried meats: possible modulation by cruciferous vegetables, glutathione S-transferase M1 , and N-acetyltransferase-2. Mutat. Res. , 19: 83-96.

Marta K and Wadysawa DA (2008a). Caffeine as a marker substrate for testing cytochrome P450 activity in human and rat. Pharmacological reports; 60(6): 789- 797.

Marta K and Wadysawa DA (2008b). The relative contribution of human cytochrome P450 isoforms to the four caffeine oxidation pathways: an in vitro comparative study with cDNA-expressed P450s including CYP2C isoforms. Biochemical pharmacology; 76(4): 543-551 .

MartinJ and Michael Fay M (2001). Cytochrome P450 drug interactions: are they clinically relevant? Aust Prescr 24: 1 0-12.

Martinez C, Ag undez JAG, Olivera M, ( ...), Benitez J (1995). Lung cancer and mutations at the polymorphic NAT2 gene locus. Pharmacogenetics, 5: 207-214.

Mashimo M, Suzuki T, Abe M, Deguchi T (1992). Molecular genotyping of N­ acetylation polymorphism to predict phenotype. Hum. Genet. 90: 139-143.

Matas N, Thygesen P, Stacey M, Risch A, Sim E (1997). Mapping MC1, MC2 and MCP, the genes for arylamine N-acetyltransferases, carcinogen metabolising enzymes on human chromosome 8p22, a region frequently deleted in tumors Cytogenet Cell Genet 77: 290-295.

May DG, Porter JA, Uetrecht JP, Wilkinson GR, Branch RA (1990). The 146 contr ibution of N-hydroxylation and acetylation to dapsone pharmacokinetics in normal subjects Clin Pharmacol Th erapeut 48: 619-627.

McDonag h EM, Boukouvala S, Aklillu E, ( ...), Klein TE (2014). PharmGKB summary: very important pharmacogene information for N-acetyltransferase 2. Pharmacogenetics and genomics; 24(8): 409 -425.

Melkersson KI, Scordo MG, Gunes A, Dahl ML (2007). Impact of CYP1A2 and CYP2D6 polymorphisms on drug metabolism and on insulin and lipid elevations and insulin resistance in clozapine-treated patients. J Clin Psychiatry. 68:697-704.

Metushi IG, Cai P, Zhu X, Nakagawa T, Uetrecht JP (2011). A fresh look at the mechanism of isoniazid-induced hepatotoxicity. Clin Pharmacol Ther. 89(6):91 1- 914.

Meyer JM and Rodvold KA (1996). Drug biotransformation by the cytochrome P- 450 enzyme system. Infect Med 13(6): 452,459,463-464,523.

Meyer UA and Zanger UM (1997). Molecular mechanisms of genetic polymorph isms of drug metabolism. Annu. Rev. Pharmaco/. Toxi co!. 37, 269-296.

Millikan RC, Pittman GS, Newman B, ( ... ), Bell DA (1998). Cigarette smoking, N­ acetyltransferases 1 and 2, and breast cancer risk. Cancer Ep idemiol. Biomark. Prev. , 7: 371 -378.

Minchin RF, Reeves PT, Teitel CH, ( ... ), lIett KF (1992). N- and O-acetylation of aromatic and heterocyclic amine carcinogens by human monomorphic and polymorphic acetyltransferases expressed in COS-1 cells. Biochem Biophys Res Commun 185: 839-844.

Minchin RF, Reeves PT, Teitel CH, ( ...), Kadlubar FF (1992). N- and O-acetylation of aromatic and heterocyclic amine carcinogens by human monomorphic and polymorphic acetyltransferases expressed in COS-1 cells. Biochem. Biophys. Res. Commun., 185: 839-844.

Mitchell JR, Thorgeirsson UP, Black M, ( . ..), Keiser HR (1975). Increased incidence of isoniazid hepatitis in rapid acetylators: possible relation to hydranize metabolites. Clin Pharmacol Ther. 18(1): 70-79.

Mizuno N, Niwa T, Yotsumoto Y, Sugiyama Y (2003). Impact of drug transporter studies on drug discovery and development. Pharmacol. Rev. 55 (3): 425-461.

Morita S, Yano M, Tasujinaka T, ( ...), Monden M (1998). Association between genetic polymorphisms of glutathione S-transferase P1 and N-acetyltransferase 2 and susceptibility to squamous-cell carcinoma of the esophagus. Int. J. Cancer, 79: 517-520.

Mortensen HM, Froment A, Lema G, ( ...), Tishkoff SA (201 1). Characterization of genetic variation and natural selection at the arylamine N-acetyltransferase genes in global human populations. Pharmacogenomics. 12(1 1): 15 147 Murata K, Hamada M, Sugimoto K, Nakano T (2007). A novel mechanism for drug­ induced liver failure: inhibition of histone acetylation by hydralazine derivatives. J Hepatol. 46(2): 322-329.

Murayama N , Soyama A, Saito Y, ( ... ), Ueno K (2004). Six novel nonsynonymous CYP1A2 gene polymorphisms: catalytic activities of the naturally occurring variant enzymes. J Pharmacol Exp Ther 308: 300-306

Nagao M, Wakabayashi K, Ushijima T, ( ...), Sugimura T (1996). Human exposure to carcinogenic heterocyclic amines and their mutational fingerprints in experimental animals Environ Health Perspect. 104: 497-501

Nakagawa K, Shindo J, Tajiri T, ( ...), Yamazaki H (2002). A population phenotyping study of three drug-metabolizing enzymes in Kyushu, Japan, with use of the caffeine test. Clin Pharmacol Th er 72: 200-208

Nakajima M, Yokoi T, Mizutani M, ( ... ), Kamataki T (1994). Phenotyping of CYP1A2 in Japanese population by analysis of caffeine urinary metabolites: absence of mutation prescribing the phenotype in the CYP1A2 gene. Cancer Epidem iology, Biomarkers & Prevention. 3: 413-421.

Nakaj ima M, Yokoi T, Mizutani M, ( ...), Kamataki T (1999). Genetic polymorphism in the 5'-flanking region of human CYP1A2 gene: effect on the CYP1A2 inducibility in humans. J Biochem. 125:803-808.

Nelson DR, Zeldin DC, Hoffman SM, ( ...), Nebert OW (2004). "Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants". Pharmacogenetics 14 (1): 1-18.

Nyberg F, Hou SM, Hemminki K, LambertBa nd Pershagen G (1998). Glutathione S-transferase M1 and N-acetyltransferase 2 genetic polymorphisms and exposure to tobacco smoke in nonsmoking and smoking lung cancer patients and population controls. Cancer Epidemiol. Biomark. Prev., 7: 875-883.

Obase Y, Shimoda T, Kawano T, ( ...), Mitsuta-Izaki K (2003). Polymorph isms in the CYP1A2 gene and theophylline metabolism in patients with asthma. Clin Pharmacol Th er. 73:468-474.

Ogawa M (1999). Biochemical, molecular genetic and ecogenetic studies of polymorphic arylamine N-acetyltransferase (NAT2) in the brain. Fukuoka Igaku Zasshi - Fukuoka Acta Me dica 90: 118-131

Okkels H, Sigsgaard T, Wolf H, Autrup H (1997). Arylamine N-acetyltransferase 1 (NAT1) and 2 (NA T2) polymorphisms in susceptibility to bladder cancer: the influence of smoking Cancer. Ep idemio/' Biomarkers Prev. , 6: 225-23 1.

Olkkola KT, Isohanni MH, Hamunen K, Neuvonen PJ (2005). The effect of erythromycin and fluvoxamine on the pharmacokinetics of intravenous lidocaine. Anesth. Ana/g. ; 100(5): 132-1356. 148

O' Neil WM, Drobitch RK, MacArthurRD, ( ...), Svensson CR (2000). Acetylator phenotype and genotype in patients infected with HIV: discordance between methods for phenotype determination and genotype. Pharmacogenetics; 10: 171- 182.

O'Neil WM, Gilfix BM, DiGirolamo A, Tsoukas CM, Wainer IW (1997). N-acetylation among HIV-positive patients and patients with AIDS: when is fast, fast and slow, slow? Clin Pharmacol Ther. 62(3): 261-271.

Ozdemir V, Kalow W, ( ...), Okey AB (2001). Treatment-resistance to clozapine in association with ultrarapid CYP1A2 activity and the C>A polymorphism in intron 1 of the CYP1A2 gene: effect of grapefruit juice and low-dose fluvoxamine. J Clin Psychopharmacol. 21: 603-607.

Paolini M, Cantelli-Forti G, Perocco P, ( ... ), Legator MS (1999). Co-carcinogenic effect of beta-carotene. (Letter) Nature 398: 760-761.

Patin E, Barreiro LB, Sabeti PC, ( ...), Ouintana-Murci L (2006a). Deciphering the ancient and complex evolutionary history of human arylamine N-acetyltransferase genes. Am J Hum Genet. 78(3): 423-436.

Patin E, Harmant C, Kidd KK, ( ...), Ouintana-Murci L (2006b). Sub-Saharan African coding sequence variation and haplotype diversity at the NAT2 gene. Hum Mutat. 27(7): 720.

Philip PA, Fitzgerald DL, Cartwright RA, Peake MD, and Rogers HJ (1988). Polymorphic N-acetylation capacity in lung cancer. Carcinogenesis (Land.), 9: 491- 493.

Philip PA, Rogers HJ, Millis RR, Rubens RD and Cartwright RA (1987). Acetylator status and its relationship to breast cancer and other diseases of the breast. Eur. J. Cancer Clin. Oneal., 23: 1701-1 706.

Poulsen HE and Loft S (1992). The impact of genetic polymorphisms in risk assessment of drugs. Arch Toxicol Supple 16: 21 1-222.

Preziosi P (2007). Isoniazid: metabolic aspects and toxicological correlates. Curr Drug Metab. 8(8): 839-851.

Probst-Hensch NM, Haile RW, Ingles SA, ( ...), Lin HJ (1995). Acetylation polymorphism and prevalence of colorectal adenomas. Cancer Res., 55: 20 1 7- 2020.

Pruitt KD, Tatusova T, Brown GR, and Maglott DR (2012). NCBI Reference Sequences (RefSe q): current status, new features and genome annotation policy. Nucleic Acids Res. 40: 0130-0135.

F186L Pucci L, Geppetti A, Maggini V, ( . . . ), Longo V (2007). CYP1A2 F21L and polymorphisms in an Italian population sample. Drug Metab Pharmacokinet. 22: 220-222. 149

Rasmussen BB, Brix TH, Kyvlk KO, Brosen K (2002). The interindividual differences in the 3-demthylation of caffeine alias CYP1A2 is determined by both genetic and environmental factors. Pharmacogenetics. 12: 473-478.

Ratain MJ, Mick R, Berezin F, ( ... ), Vogelzang NJ (1993). Phase I study of amonafide dosing based on acetylator phenotype. Cancer Res; 53: 2304-2308

Ratain MJ, Mick R, Berezin F, Janisch L, ( ...) , Williams SF (1991). Paradoxical relationship between acetylator phenotype and amonafide toxicity. Clin Pharmacal Th erapeut; 50: 573-579

Ratain MJ, Mick R, Janisch L, ( ... ), Vogelzang NJ (1996). Individualized dosing of amonafide based on a pharmacodynamic model incorporating acetylator phenotype and gender. Pharmacogenetics; 6: 93-1 01

Ratain MJ, Rosner G, Allen SL, ( ...) Henderson IC (1995). Population pharmacodynamic study of amonafide: a Cancer and Leukemia Group B study. J Clin Oncol; 13: 741-747

Reiling MV, Lin JS, Ayers GO, Evans WE (1992) Racial and gender differences in N-acetyltransferase, xanthine oxidase, and CYP1A2 activities. Clin Pharmacal Ther. 52: 643-658.

Rihs HP, John A, Scherenberg M, Seidel A, Bruning T (2007). Concordance between the deduced acetylation status generated by high-speed: real-time PCR based NAT2 genotyping of seven single nucleotide polymorphisms and human NAT2 phenotypes determined by a caffeine assay. Clin Chim Acta. 376(1-2): 240- 243.

RobertK, Daryl K, Peter A and Victor W (2003). Harper's illustrated biochemistry (26th ed.). New York; London: McGraw-Hili. Page 14 -72.

Roberts-Thomson IC, Ryan P, Khoo KK, ( . ..), Butler RN (1996). Diet, acetylator phenotype, and risk of colorectal neoplasia. Lancet, 347: 1372-1 374.

Rodriguez JW, Kirlin WG, Ferguson RJ, ( ... ) , Hein OW (1993). Human acetylatorgenotype: relationship to colorectal cancer incidence and arylamine N­ acetyltransferaseexpression in colon cytosol. Arch. Toxicol. , 67: 445-452.

Roots me, Drakoulis N, Ploch M, ( ... ), Koch M (1988). Debrisoquine hydroxylation phenotype, acetylation phenotypes, and ABO blood groups as genetic host factors of lung cancer risk. Klin. Wochenshr. , 66 (Suppl XI): 87-97.

Ruiz JD, Martinez C, Anderson K, ( ... ), Ag undez JA (2012).The differential effect of NAT2 variant alleles permits refinement in phenotype inference and identifies a very slow acetylation genotype. PLoS One. 7(9):e44629.

Runge 0, Kohler C, Kostrubsky V, Janger 0, Lehmann T, Runge 0, May U, Stolz S, Strom S, Fleig Wand Michalopoulos G (2000). Induction of Cytochrome P450 (CYP)1A1, CYP1A2, and CYP3A4 but Not of CYP2C9, CYP2C19, Multidrug Resistance (MDR-1) and Multidrug Resistance Associated Protein (MRP-1) by 150

Prototypical Inducers in Human Hepatocytes. Biochemical and Biophysical Research Communications; Vol. 273:1 Pag. 333-341.

Sabbagh A. Langaney A, Darlu P, ( ... ), Poloni ES (2008). Worldwide distribution of NAT2 diversity: implications for NAT2 evolutionary history. BMC Genet. 9: 21.

Sachse C, Bhambra U, Smith G, ( ... ), Scollay J (2003). Polymorphisms in the cytochrome P450 CYP1A2 gene (CYP1A2) in colorectal cancer patients and controls: allele frequencies, linkage disequilibrium and influence on caffeine metabolism. Br J Clin Pharmacol. 55: 68-76.

Sachse C, Brockmoller J, Bauer S, Roots I (1999). Functional significance of a C­ to-A polymorphism in intron 1 of the cytochrome P450 CYP1A2 gene tested with caffeine. Brit. J. Clin. Pharm. 47: 445-449.

Saito Y, Hanioka N, Maekawa K, ( ...), Nakamura R (2005). Functional analysis of three CYP1A2 variants found in a Japanese population. Drug Metab Dispos 33: 1905-1910

Sardas S, Cok I, Sardas OS, IIhan 0 and Karakaya AE (1990). PolymorphicN­ acetylation capacity in breast cancer patients. Int. J. Cancer, 46: 1138-1 139.

Sarich T, Kalhorn T, Magee S, ( ...), Wright J (1997). The effect of omeprazole pretreatment on acetaminophen metabolism in rapid and slow metabolizers of S­ mephenytoin. Clin. Pharmacol. Ther.; 62(1): 21-28.

Selinski S, Blaszkewicz M, Agundez JA, ( ...), Hengstler JG (2013a). Clarifying haplotype ambiguity of NAT2 in multi-national cohorts. Front Biosci (Schol Ed) 5:672-684.

Selinski S, Blaszkewicz M, Ickstadt K, Hengstler JG, Golka K (2013b). Refinement of the prediction of N-acetyltransferase 2 (NAT2) phenotypes with respect to enzyme activity and urinary bladder cancer risk. Arch Toxico!. 87(12): 2129-2139.

Shear NH, Spielberg SP, Grant OM, Tang BK, Kalow W (1986). Differences in metabolism of sulfonamides predisposing to idiosyncratic toxicity. Ann Intern Med. 105: 179-184

Shepherd AM, Ludden TM, McNay JL, Lin MS (1980). Hydralazine kinetics after single and repeated oral doses Clin Pharmacol Th erapeut. ; 28: 804-811.

Shibuta K, Nakashima T, Abe M, ( ... ), Suzuki T (1994). Molecular genotyping for N­ acetylation polymorphism in Japanese patients with colorectal cancer. Cancer (Phila.),7 4: 3108-3112.

Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP (1994). Interindividual variations in human liver cytochrome P450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals. J Pharmacol Exp Ther. 270: 414-423.

Shimizu T, Tateishi T, Hatano M, Fujii-Kuriyama Y (1991). Probing the role of Iysines and arginines in the catalytic function of cytochrome P450d by site-directed 151 mutagenesis. Interaction with NADPH-cytochrome P450 reductase. J BioI Chem. 266(6): 3372-3375

Sim E, Fakis G, Laurieri N, Boukouvala S. ( 2012). Arylamine N-acetyltransferases­ -from drug metabolism and pharmacogenetics to identification of novel targets for pharmacological intervention. Advances in pharmacology; 63: 169-205.

Sim E, Lack N, Wang C,( ...) Kawamura A (2008) Arylamine N-acetyltransferases: structural and functional implications of polymorphisms. Toxicology; 254(3): 170- 183.

Sinclair JC, Sandy J, Delgoda R, ( ...) Noble ME (2000) Structure of arylamine N­ acetyltransferase reveals a catalytic triad. Nat Struct BioI; 7: 560-564

Skarke C, Kirchhof A, Geisslinger G, Lotsch J (2005). Rapid genotyping for relevant CYP1A2 alleles by pyrosequencing. Eur J Clin Pharmacol.; 61: 887-892.

Skog K, Steineck G, Augustsson K and Jugerstad M (1995). Effect of cooking temperature on the formation of heterocyclic amines in fried meat products and pan residues. Carcinogenesis (Lond.), 16: 861 -867,

Smelt VA, Mardon HJ and Sim E (1998) Placental expression of arylamine N­ acetyltransferases: evidence for linkage disequilibrium between NAT1 *1 0 andNAT2*4 alleles of the two human N-acetyltransferase loci NAT1 and NAT2. Pharmacol. Toxicol., 83: 149-157,

Smith CJ, Livingston SO, Doolittle OJ (1997). An international literature survey of 'IARC Group I carcinogens' reported in mainstream cigarette smoke Food & Chem Toxico!.; 35: 1107-1 130

Soejima M, Kawaguchi Y, Hara M, Kamataki N (2008). Prospective study of the association between NAT2 gene haplotypes and severe adverse events with sulfasalazine therapy in patients with rheumatoid arthritis. J Rheumatol. ; 35(4): 724.

Soejima M, Sugiura T, Kawaguchi Y, C ...), Kamatani N (2007). Association of the diplotype configuration at the N-acetyltransferase 2 gene with adverse events with co-trimoxazole in Japanese patients with systemic lupus erythematosus. Arthritis Res Ther. 9(2): R23.

Soyama A, Saito Y, Hanioka N, ( ... ), Kamakura S (2005). Single nucleotide polymorphisms and haplotypes of CYP1A2 in a Japanese population. Drug Metab Pharmacokinet. 20: 24-33.

Spielberg SP (1996). N-acetyltransferases: pharmacogenetics and clinical consequences of polymorphic drug metabolism J Pharmacokinet Biopharmaceut 24: 509-519

Spinasse LB, Santos AR, Suffys PN, ( ... ) Salles GF (2014). Different phenotypes of the NA T2 gene influences hydralazine antihypertensive response in patients with 152 resistant hypertension. Pharmacogenomics; 15(2): 169-1 78.

Spurr NK, Gough AC, Chinegwundoh FI and Smith CAD (1995). Polymorphisms in drug-metabolizing enzymes as modifiers of cancer risk. Clin. Chem. , 41 : 1864- 1869.

Sram RJ and Binkova B (2000). Molecular epidemiology studies on occupational and environmental exposure to mutagens and carcinogens. Environ Health Perspect ; 108: 57-70

Stanley LA and Sim E (2008). Update on the pharmacogenetics of NATs: structural considerations. Pharmacogenomics; 9(1 1): 1673-1693.,

Starheim K, Gevaert K, Arnesen T (2012). Protein N-terminal acetyltransferases: when the start matters. Trends in Biochemical Sciences 37 (4): 152-1 61.

Stephens M, Smith NJ and Donnelly P (2001). A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 68, 978-989.

Stone EM, Williams JA, Grover PL, Gusterson BA, and Phillips DH (1998). Interindividual variation in the metabolic activation of heterocyclic amines and their N-hydroxy derivatives in primary cultures of human mammary epithelial cells. Carcinogenesis (Lond.), 19: 873-879.

Sulem P, Gudbjartsson DF, Geller F, ( .. . ) Aben KK (201 1). Sequence variants at CYP1A1 -CYP1A2 and AHR associate with coffee consumption. Hum Mol Genet. ; 20: 2071-2077.

Suzuki H , Morris JS, Li Y, ( ...) Liu J (2008). Interaction of the cytochrome P4501A2, SULT1A 1 and NAT gene polymorphisms with smoking and dietary mutagen intake in modification of the risk of pancreatic cancer. Carcinogenesis ; 29: 1184-1 191

Takata K, Saruwatari J, Nakada N, ( ...) Tanaka F (2006). Phenotype-genotype analysis of CYP1A2 in Japanese patients receiving oral theophylline therapy. Eur J Clin Pharmacol. ;62: 23-28.

Tan EK, Khajavi M, Thornby JI, ( . . . ), Ashizawa T (2000). Variability and validity of polymorphism association studies in Parkinson's disease. Neurology; 55(4): 533- 538.

Tang B, Zhou Y, Kadar D and Kalow W (1994). Caffeine as a probe for CYP1A2 activity: potential influence of renal factors on urinary phenotypic trait measurements. Pharmacogenetics; 4(3): 117-124.

Thorn CF, Aklillu E, McDonagh EM, ( ...) Altman RB (2012). PharmGKB summary: caffeine pathway. Pharmacogenet Genomics.; 22(5): 389-395.

Tiwari AK, Deshpande SN, Rao AR, ( ...) Shriharsh V (2005). Genetic susceptibility to tardive dyskinesia in chronic schizophrenia subjects: I. Association of CYP1A2 153 gene polymorphism. Pharmacogenomics J. ; 5: 60-69.

Tostmann A, Boeree MJ, Aarnoutse RE, ( ...), Oekhuijzen R (2008). Antituberculosis drug-induced hepatotoxicity: concise up-to-date review. J Gastroenterol Hepatol. ; 23(2): 192-202.

Trepanier LA, Ray K, Winand NJ, ( ...) Cribb AE (1997). Cytosolic arylamine N­ acetyltransferase (NAT) deficiency in the dog and other canids due to an absence of NAT genes. Biochem Pharmacol 54: 73-80.

Turesky RJ, Lang NP, Butler MA, Teitel CH and Kadlubar FF (1991). Metabolic activation of carci nogenic heterocyclic aromatic amines by human liver and colon. Carcinogenesis (Lond.), 12: 1839-1845.

Ueda R, Iketaki H, Nagata K, ( ...) Kusano K (2006). A common regulatory region functions bidirectionally in transcriptional activation of the human CYP1 A 1 and CYP1A2 genes. Mol Pharmacol. ; 69: 1924-1930.

Van OP, Hole K, Pimenta-Marques A, ( . . . ), Arnesen T (201 1). Nat2 Contributes to an Evolutionary Shift in Protein N-Terminal Acetylation and Is Importantfor Normal Chromosome Segregation. PLoS Genetics 7 (7): e1002169.

Vander HL, Peeters PH, Hein OW, ( . . . ), Bueno OM (2003). NAT2 slow acetylation and STM1 null genotypes may increase postmenopausal breast cancer risk in long­ term smoking women. Pharmacogenetics; 13: 399-407.

Vats is KP, Weber WW,Bell OA, ( ... ), Yamazoe Y (1995). Nomenclature for N­ acetyltransferases. Pharmacogenetics, 5: 1-17.

Vercruysse A (1997). "Toxicologie." Cursus 2de graad apotheker VUB ;pp. 17.

Wadelius M, Autrup JL, Stubbins MJ, (. . . ), Rane A (1999). Polymorphisms in NAT2, CYP206, CYP2C 19, and GSTP1 and their association with prostate cancer. Pharmacogenetics, 9: 333-340,

Walraven JM, Zang Y, Trent JO, Hein OW (2008). Structure/function evaluations of single nucleotide polymorphisms in human N-acetyltransferase 2. Curr Drug Metab.; 9(6): 471 -486.

Wang B and Zhou SF (2009). Synthetic and natural compounds that interact with human cytochrome P450 1A2 and implications in drug development. Curr Med Chem.; 16(31): 4066-4218.

Wang L, Tang W, Chen S, ( ...), Gu H (2014). N-acetyltransferase 2 polymorphisms and risk of esophageal cancer in a Chinese population. PLoS One. 19; 9(2): e87783.

Wang Z, Chen M, Zhu L, Yu L, Zeng S, Xiang M, Zhou Q (2015). Pharmacokinetic drug interactions with clopidogrel: updated review and risk management in combination therapy. Ther Clin Risk Manag.; 11: 449-467. 154

Waxman OJ and Chang TK (2006). Use of 7 -ethoxycoumarin to monitor multiple enzymes in the human CYP1, CYP2, and CYP3 families. Methods Mol BioI .; 320: 153-156.

Weber WW and Hein OW (1985). N-acetylation pharmacogenetics. Pharmacol Rev.; 37(1): 25-79.

Webster OJ, Flook 0, Jenkins J, Hutchings A and Routledge PA (1989). Drug acetylation in breast cancer. Br. J. Cancer, 60: 236-237.

Welfare MR, Cooper J, Bassendine MF ( ...), Daly AK (1997). Relationship between acetylator status, smoking, and diet and colorectal cancer risk in the north-east of England, Carcinogenesis 18: 1351-1 354

Wohlleb JC, Hunter CF, Blass B,(. ..) , Lang NP (1990). Aromatic amine acetyltransferase as a marker for colorectal cancer: environmental and demographic associations Int J Cancer; 46: 22-30

Wolkenstein P, Loriot MA, Aractingi S,(. ..), Chosidow 0 (2000). Prospective evaluation of detoxification pathways as markers of cutaneous adverse reactions to sulphonamides in AIDS. Pharmacogenetics; 1 0(9): 821-828.

Woolhouse NM, Qureshi MM, Bastaki SM ( ...), Bayoumi RA (1997). Polymorphic N-acetyltransferase (NAT2) genotyping of Emiratis. Pharmacogenetics; 7(1): 73-82.

Yamasaki Y, leiri I, Kusuhara H,(.. ) , Sugiyama Y (2008). Pharmacogenetic characterization of sulfasalazine disposition based on NAT2 and ABCG2 (BCRP) gene polymorphisms in humans. Clin Pharmacol Ther. ;84(1): 95-103.

Yuan R, Madani S, Wei XX, Reynolds K, Huang SM (2002). Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab Dispos. ;30:131 1-1319.

Yuliwulandari R, Sachrowardi Q, Nishida N ( ...) , Tokunaga K (2008). Polymorph isms of promoter and coding regions of the arylamine N­ acetyltransferase 2 (NAT2) gene in the Indonesian population: proposal for a new nomenclature. J. Hum. Genet. 53: 201-209.

Zanger UM, Turpeinen M, Klein K, Schwab M (2008). Functional pharmacogenetics/ genomics of human cytochromes P450 involved in drug biotransformation. Anal Bioanal Chem.; 392: 1093-1 108

Zgheib NK, Shamseddine AA, Geryess E, ( ... ), EI-Saghir NS (2013). Genetic polymorphisms of CYP2E 1, GST, and NAT2 enzymes are not associated with risk of breast cancer in a sample of Lebanese women. Mutat Res. ; 747-748: 40-47.

Zhang L, Xiang Z, Hao R, Li R, Zhu Y (2014). N-acetyltransferase 2 genetic variants confer the susceptibility to head and neck carcinoma: evidence from 23 case-control studies. Tumour BioI; 35(4): 3585-3595.

Zhao B, Seow A, Lee EJ, Lee HP (2000). Correlation between acetylation 155 phenotype and genotype in Chinese women. Eur J Clin Pharmacal; 56(9-10): 689- 692.

Zhou H, Jasephy PO, Kim 0, Guengerich FP (2004). Functional characterization of four allelic variants of human cytochrome P450 1A2. Arch Biochem Biophys ; 422: 23-30.

Zhou SF, Di YM, Chan E, ( ... ), Duan W. (2008). Clinical pharmacogenetics and potential application in personalized medicine. Curr Drug Metab.; 9: 738-784.

Zhou SF, Koh HL, Gao YH, Gong ZY, Lee EJD (2004). Herbal bioactivation: the good, the bad and the ugly. Life Sci; 74: 935-968.

Zhou SF, Liu JP, Chowbay B (2009a). Polymorphism of human cytochrome P450 enzymes and its clinical impact. Drug Metab Rev. ; 41: 89-295.

Zhou SF, Yang LP, Zhou ZW, Liu YH, Chan E (2009b). Insights into the substrate specificity, inhibitors, regulation, and polymorph isms and the clinical impact of human cytochrome P450 1A2. AAPS J. 11: 481-494.

Zielinska E, Niewiarowski W, Bodalski J (1998). The arylamine N-acetyltransferase (NAT2) polymorphism and the risk of adverse reactions to co-trimoxazole in children. Eur J Clin Pharmacal. ; 54(9-10): 779-85. 156

List of Publications

Papers:

• Mohammad M. AI-Ahmad, Naheed Amir, Subramanian Dhanasekaran, Anne John, Yousef M. Abdulrazzaq, Bassam R. Ali & Salim Bastaki. Polymorphic N-acetyltransferase (NAT2) Phenotyping and Genotyping among Emiratis. (submitted to Annals of Human Genetics - AHG-MS-16-01 99)

• Salim M. A. Bastaki, Mohammed Majed AI Ahmed , Ahmed AI Zaabi, Naheed Amir, Ernest Adeghate. Effect of turmeric on colon histology, body weight, ulcer, IL-23, MPO and glutathione in acetic-acid-induced inflammatory bowel disease in rats. BMC Complement Altern Med. 2016; 16: 72. Published online 2016 Feb 23. doi: 10.1186/s12906-016-1057-5. PMCID: PMC4763431. https :llwww.ncbi.nlm.nih.gov/pm c/articles/PMC4763431

• Chandranath IR, Bastaki SMA, AIAhmad MM, MA, Singh J "Protective Mechanisms of the Gastrointestinal Mucosa: A Review". Research Trends: Current Topic in Pharmacology, volume 18, page 1-28. 2014. http://www.researchtrends.netltia/title issue.asp?id=11 &in=0&vn=18 157

Appendices

Appendix 1: Research Ethical Approval

Appro the Study

DlI-IHEC No. 21,.,0 9 - 12· ·tyl on henofyp og'g no pin 01

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Appendix 2: Research Ethical Approval - extended

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Appendix 5: Dubai Education Zone Ethical Approval

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Appendix 6: RAK Education Zone Ethical Approval

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Appendix 8: Data collection fo rm

Acetylation Phenotyping and Genotyping Study

Name: no: ------10 ------

Nationality:

Are father and mother related? LJLJ If the answer to above is yes what is the relation?

1. Father's father and mother's father are brothers. 2. Father's father and mother's mother are brother and sister. 3. Father's father and mother's mother are sisters. 4. Father's father and mother's father are sister and brother. 5. Father's father and mother's grandfather are brothers. 6. Father's father and mother's grandmother are brother and sister. 7. Father's father and mother's grandmother are sisters. 8. Father's father and mother's grandfather are sister and brother.

9. Other close relation ------10. Same tribe

Emirate

Age Sex EJ I Female I

Smoker 165

Appendix 8: Data collection fo rm- continue

Existence of any medical condition

If answer to above is yes, what is (are) the medical condition (s)?

Any mild, moderate or severe reaction to any drug?

If the answer to the above question is yes, what is the name of the drug?

If the answer to the above question is yes, what was the reaction that occurred? 166 Appendix 8: Data collection fo rm - continue

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