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Cytochrome P450 2C19 Polymorphisms and
Clopidogrel Management in Patients with
Coronary Artery Disease in Gaza Strip-Palestine
مرضي في Cytochrome P450 2C19لجيه ط المتعددةاالومب
فلسطيه -في قطبع غزة Clopidogrelالشراييه التبجيت المعبلجيه بعقبر
By
Ibrahim Raji Al-Astal
Supervised by
Prof.Dr. Maged M.Yassin Dr. Basim M. Ayesh
Professor of Physiology Associate professor of genetics
and molecular biology
A thesis submitted in partial fulfilment
of the requirements for the degree of
Master of Medical Technology
May / 2016
زةــغ – تــالميــــــت اإلســـــــــبمعـالج
والدراسبث العليبشئون البحث العلمي
ــــــلومــــــــــــــــــــــــــت العـــليــــــك
تــــــــــــــتحبليـــــــــــل طبيمبجستير
The Islamic University-Gaza
Research and Postgraduate Affairs
Faculty of Science
Master of Medical Technology
II
Abstract
Background: Platelets play a central role in the pathophysiology of the acute
coronary syndromes following percutaneous coronary intervention (PCI).
Clopidogrel is an oral thienopyridine derivative capable of inhibiting platelet
activation. Clopidogrel is a prodrug that is converted into an active drug by the
hepatic cytochrome CYP2C19. The CYP2C19*2 and the CYP2C19*3 polymorphic
alleles are considered to be important loss-of-function alleles resulting in diminished
response to Clopidogrel.
Objective: The aim of this study was to determine the allelic frequency of CYP2C19
wild type allele (CYP2C19*1) and its loss of function variants (alleles *2 and *3),
and their role in recurrence of cardiovascular disease in PCI patients receiving
Clopidogrel in Gaza strip.
Methods: This study is cross sectional study with convenience sample. Whole blood
samples were collected from 110 patients undergoing PCI under clopidogrel therapy.
The frequency of CYP2C19 alleles was determined by the polymerase chain reaction
with restriction fragment length polymorphism (PCR-RFLP).
Results: The frequency of CYP2C19*1, *2 and *3 alleles was 82.3%, 15.5% and
2.3% respectively. Genotyping analysis showed that, 67.3% were homozygotes for
CYP2C19*1, 27.3 % were *1/*2, 2.7% with *1/*3 genotype, 1.8% were *2/*3 and
0.9% were *2/*2. These frequencies were comparable to those of other Caucasian
populations. According to this study the poor metabolizers (PM) phenotype
frequency was 2.7 %, which is in the same range reported in Caucasians (2 to 5%)
and lower than Oriental populations 13-23%. A strong significant relation was found
between stent restenosis and carrying the variant allele CYP2C19*2 (P= 0.001). On
the other hand, there was no significant relation between stent restenosis and carrying
the variant allele CY2C19*3 (p = 0.324).
Conclusion: The CYP2C19*2 and CYP2C19*3 alleles genotyping should be
included in the workup of patient considered for Clopidogrel therapy to avoid in-
stent restenosis after PCI procedure.
Keywords: Clopidogrel; CYP2C19; Coronary artery disease; percutaneous coronary
intervention.
III
Abstract in Arabic
الممخص
المرضي لمتالزمة الشريان التاجي الحادة بعد عممية رأب الوعاء التطورالصفائح الدموية تمعب دورا رئيسا في مقدمة:
الي انزيم يحتاج Clopidogrel عقار الصفائح الدموية. نشاطعمي تثبيط Clopidogrelيعمل عقار (. PCIالتاجي )
CYP2C19 في الكبد. ومن بين االنماط األليمية اختالف نيوكموتيدة وحيدة لتنشيطوSNP) )لمجين CYP2C19 ،
.لفاعميتوالدواء فقداناحد اسباب CYP2C19*3)النمط الثالث )و ( (CYP2C19*2لمنمط الثاني تغير الشكل الجيني
في CYP2C19*3و CYP2C19*1 ،CYP2C19*2االنماط االليمية التالية معدل تكرارتحديد هدف الدراسة:
.PCIبعد عممية Clopidogrelقمب لممرضي المعالجين بواسطة لفي تجدد مشاكل ا SNPقطاع غزة وتحديد دور
ويعالجون بعقار PCI من المرضي المذين خضعوا لعممية 111عينات دم خام تم جمعيا من المواد المستخدمة:
clopidogrel طريقاستخدام ب وتم فحص االختالفات الجينية ليم PCR-RFLP (الحامض النووي إكثار جزء من
.)قاطعة متخصصة إنزيماتبواسطة قطعياالديؤكسي ريبوزي ومن ثم
كانت بالترتيب عمي النحو CYP2C19لمجين 3و 2، 1اظيرت الدراسة ان نسبة وجود الشكل الجيني رقم النتائج:
% من المرضي متماثل الزيجوت لمجين 3..3%(. واظير تحميل االنماط الجينية ان 2.3% و 11.1%، 32.3التالي)
% متغاير..2(، و (2*/1*% متغاير الزيجوت لمنمط الجيني االول مع الثاني 3..2(، و CYP2C19*1السميم )
(، 3*/2*% متخالف الزيجوت لمنمط الثاني مع الثالث)1.3(، و 3*/*1الزيجوت لمنمط الجيني االول مع الثالث )
(، ولم تظير الدراسة وجود نمط متماثل الزيجوت لمنمط الثالث 2*/2*% متماثل الزيجوت لمنمط الثاني )1.0و
و% وىذه النسب..2كانت (PM)في كفاءة االنزيم شديد (. وجد ان نسبة المرضي المذين يعانون من نقص3*/3*)
مشابية لمدراسات التي اجريت عمي العرق القوقازي. وقد وجدت عالقة ذات داللة احصائية بين الشكل الجيني
CYP2C19*2 و عممية اعادة تضيق الشريان (P= 0.001 وقد لوحظ عدم وجود عالقة ذات داللة احصائية )
CYP2C19*3 (p=0.324). بالنسبة لمشكل الجيني
في عممية متابعة يأخذ بعين االعتباريجب ان CYP2C19*3و CYP2C19*2 تحديد النماط االليمية الخالصة:
PCI.لتفادي حدوث تضيق في الشريان التاجي بعد عممية Clopidogrelالمرضي المذين سيتم اعطائيم عقار
غزة. امراض الشرايين التاجية، الجيني، ، النمطرأب الوعاء التاجي كممات مفتاحية:
IV
Dedication
I dedicate this work with my deep love to
My Parents
For their endless love, support, inspiration and continuous sacrifices
My Wife
Who has been a constant source of support and encouragement
My Lovely Kids
Tala, Ahmed, Mahmoud and ….
My Brothers and Sisters
For their encouragements
My Friends and Colleagues
V
Acknowledgements
There are a number of people to whom I express my Sincere gratitude without whom
this thesis might not have been structured, and to whom I am greatly thankful.
Dr. Basim Ayesh, the ideal thesis supervisor. His sage advice, insightful criticisms,
and patient encouragement aided the writing of this thesis in innumerable ways. I
thank Dr. Maged Yassin Whose steadfast support of this research was greatly needed
and deeply appreciated.
My deep and sincere appreciations to my colleagues in the laboratory of the
European Gaza Hospital for their support, generous assistance and valuable
comments.
To all my friends, thank you for the sense of belonging and fraternity that you
fostered.
My special deep and sincere gratitude to my parents, wife, brothers and sisters for
their encouragement, continuous support and help.
Finally, a hearty thanks to my family for their understanding, support, and sacrifice.
Without their guidance, help and patience, I would have never been able to
accomplish the work of this thesis.
VI
Table of Contents
Declaration .................................................................................................................... I
Abstract ........................................................................................................................ II
Abstract in Arabic ...................................................................................................... III
Dedication .................................................................................................................. IV
Acknowledgements ...................................................................................................... V
Table of Contents ....................................................................................................... VI
List of Tables ............................................................................................................. IX
List of Figuers .............................................................................................................. X
List of Abbrevation .................................................................................................... XI
Chapter (1): Introduction ......................................................................................... 1 1.1 Overview ............................................................................................................ 1
1.2 General objective ............................................................................................... 3
1.3 Specific objectives ............................................................................................. 3
1.4 Significance ....................................................................................................... 3
Chapter (2):Literturer Review ................................................................................. 4 2.1 Heart and coronary arteries ................................................................................ 4
2.2 Definition of coronary artery disease ................................................................ 5
2.3 Mortality rate of Cardiovascular diseases .......................................................... 5
2.4 Pathophysiology of coronary artery disease ...................................................... 6
2.5 Risk factor of coronary artery disease ............................................................... 7
2.6 Cytochrome P450 .............................................................................................. 7
2.6.1 Definition ........................................................................................................ 7
2.6.2 Nomenclature of CYP ..................................................................................... 7
2.6.3 Classification of CYP ..................................................................................... 7
2.6.4 Function of CYP ............................................................................................. 8
2.7 Cytochrome P450 2C19 ..................................................................................... 8
2.7.1 Definition and function ................................................................................... 8
2.7.2 CYP2C19 gene ............................................................................................... 8
2.7.3 Expression of CYP2C19 ................................................................................. 9
2.7.4 Polymorphism of CYP2C19 ........................................................................... 9
2.7.4.1 The CYP2C19*2 ........................................................................................ 10
2.7.4.2 The CYP2C19*3 ........................................................................................ 10
2.7.4.3 Other CYP2C19 variants that encode reduced or unknown enzymatic
activity ................................................................................................................ 10
2.7.4.4 CYP2C19 Variants that encode increased enzymatic activity................... 11
2.7.5 Impact of CYP2C19 (polymorphism) genotype on drug metabolism .......... 11
2.8 Management of coronary artery disease .......................................................... 12
2.8.1 Coronary revascularization ........................................................................... 12
2.8.1.1 Balloon angioplasty ................................................................................... 12
2.8.1.2 Percutaneous coronary intervention (PCI) ................................................. 13
2.8.2 Antiplatelet therapy ...................................................................................... 13
2.8.2.1 P2Y12 receptor antagonists ......................................................................... 14
2.9 Clopidogrel ...................................................................................................... 14
2.9.1 Definition and structure ................................................................................ 14
2.9.2 Pharmacokinetics of clopidogrel .................................................................. 15
2.9.3 Pharmacodynamic of clopidogrel ................................................................. 15
VII
2.10 Prevalence and relation of CYP2C19*2 and *3 alleles to the platelet
inhibition by clopidogrel .................................................................................... 17
Chapter (3): Materials and Methodes .................................................................... 21 3.1. Study design .................................................................................................... 21
3.2. Study population ............................................................................................. 21
3.3. Sample size ..................................................................................................... 21
3.4. Ethical consideration ...................................................................................... 21
3.5. Inclusion criteria ............................................................................................. 22
3.6. Exclusion criteria ............................................................................................ 22
3.7. Materials ......................................................................................................... 22
3.7.1. Instruments .................................................................................................. 22
3.7.2. List of reagents and chemicals ..................................................................... 23
3.7.3. Primers used in this study ............................................................................ 23
3.7.4. Disposables .................................................................................................. 24
3.8. Methods .......................................................................................................... 24
3.8.1. Data collection ............................................................................................. 24
3.8.2. Blood samples collection ............................................................................. 24
3.9. Molecular analysis .......................................................................................... 25
3.9.1. Extraction and purification of genomic DNA ............................................. 25
3.9.2. Primers reconstitution .................................................................................. 27
3.10. Detection of CYP2C19*2 and CYP2C19*3 polymorphisms by PCR-RFLP 27
3.10.1. Polymerase Chain Reaction (PCR) for CYP2C19*2 ................................. 27
3.10.2. Restriction fragment length polymorphism (RFLP) CYP2C19*2 PCR
product by Sma1 restriction enzyme .................................................................. 28
3.10.3. Polymerase chain reaction (PCR) for CYP2C19*3 ................................... 29
3.10.4. Restriction fragment length polymorphism (RFLP) of CYP2C19*3 PCR
product by BamH1 restriction enzyme ............................................................... 30
3.11. Interpretation of CYP2C19 alleles ................................................................ 30
3.12. Quality control .............................................................................................. 31
3.13. Statistical analysis ......................................................................................... 31
Chapter (4): Results ................................................................................................. 32 4.1. Characteristics of the study population: ......................................................... 32
4.1.1. Personal characteristics ................................................................................ 32
4.1.2 Clinical characteristics .................................................................................. 33
4.1.2.1 Cardiac and other chronic diseases ............................................................ 33
4.1.2.2. Complete blood count of the study population ......................................... 33
4.1.2.3 Angiographic outcomes ............................................................................. 33
4.2. PCR and RFLP results for CYP2C19 ............................................................. 34
4.2.1. CYP2C19*2 PCR product gel electrophoresis ............................................ 34
4.2.2. CYP2C19*2 restriction analysis gel electrophoresis .................................. 35
4.2.3. CYP2C19*3 PCR product gel electrophoresis ............................................ 35
4.2.4. CYP2C19*3 restriction analysis gel electrophoresis ................................... 36
4.3. The CYP2C19 Genotyping results ................................................................. 36
4.3.1. Genotyping frequency of CYP2C19*2 allele .............................................. 36
4.3.2. Genotyping frequency of CYP2C19*3 allele .............................................. 37
4.3.3. Overall genotype distribution of CYP2C19 ................................................ 37
4.3.4. Distribution of CYP2C19 genotypes frequency by gender .......................... 38
4.3.5 The CYP2C19 genotype and predicted phenotype ....................................... 39
VIII
4.4 Stent restenosis ................................................................................................ 39
4.4.1. Impact of CYP2C19 polymorphism on stent restenosis .............................. 40
4.4.1.1. Impact of CYP2C19*2 mutation on stent restenosis ................................ 40
4.4.1.2 Impact of CYP2C19*3 mutation on stent restenosis ................................. 40
4.4.2. Relationship between restenosis and hypertension ...................................... 41
4.4.3. Relation between restenosis and Diabetes mellitus ..................................... 41
4.4.4. Relation between restenosis and age ........................................................... 41
4.4.5. Relation between restenosis and gender ...................................................... 42
4.4.6 Relation between restenosis and body mass index (BMI) ............................ 42
4.4.7 Relation between restenosis and family history of CAD .............................. 43
4.4.8 Relation between restenosis and Segment of coronary legion ..................... 43
Chapter (5): Discussion ........................................................................................... 44 Discussion .............................................................................................................. 44
Chapter (6): Conclusions and Recommendations................................................. 49 6.1. Conclusions ..................................................................................................... 49
6.2. Recommendations ........................................................................................... 50
The References List ................................................................................................. 51
Annex (1): Approval of Helsinki committee .......................................................... 65
Annex (2): Approval of MOH for sample collection ............................................ 66
Annex (3): Data collection ....................................................................................... 67
IX
List of Tables
Table (2.1): Commonly CYP2C19 variant alleles and their effect on CYP2C19
protein. ................................................................................................................... 11
Table (3.1): List of laboratory instruments ............................................................... 22
Table (3.2): Lists the chemicals and reagents ........................................................... 23
Table (3.3): Nucleotide sequence of the PCR primers ............................................. 23
Table (3.4): List of disposables ................................................................................. 24
Table (3.5): Wizard genomic DNA purification kit solutions .................................. 26
Table (3.6): Materials that should be supplied by the user ....................................... 26
Table (3.7): PCR components for amplification of CYP2C19*2 ............................. 28
Table (3.8): Thermocycler program for PCR amplification of the CYP2C19*2 and
CYP2C19*3 ........................................................................................................... 28
Table (3.9): The enzymatic digestion components of amplified CYP2C19*2 ......... 29
Table (3.10): PCR components for amplification of CYP2C19*3 ........................... 29
Table (3.11): The enzymatic digestion components of amplified CYP2C19*3 ....... 30
Table (3.12): Interpretation of CYP2C19 alleles ...................................................... 30
Table (3.13): Predicted metabolism phenotypes for CYP2C19 based on example
genotypes................................................................................................................ 31
Table (4.1): Personal characteristics of the study population ................................... 32
Table (4.2): Comorbid chronic disease in the study population ............................... 33
Table (4.3): Complete blood count of the study population ..................................... 33
Table (4.4): Lesion location and type of instilled stent ............................................ 34
Table (4.5): Genotyping Frequency of CYP2C19*2 allele ...................................... 37
Table (4.6): Frequency of genotypes of CYP2C19*3 allele ..................................... 37
Table (4.7): Genotype distribution of CYP2C19 ...................................................... 38
Table (4.8): Distribution of CYP2C19 genotypes by gender ................................... 38
Table (4.9): Distribution of restenosis cases by the CYP2C19*2 allele genotype ... 40
Table (4.10): Distribution of restenosis cases according to CYP2C19*3 genotype . 41
Table (4.11): Distribution of stent restenosis patients by hypertension .................... 41
Table (4.12): Distribution of stent restenosis patients by diabetes mellitus ............. 41
Table (4.13): Distribution of stent restenosis patients by age ................................... 42
Table (4.14): Distribution of stent restenosis patients by gender ............................. 42
Table (4.15): Distribution of stent restenosis patients by BMI ................................. 42
Table (4.16): Distribution of stent restenosis patients by Family history of heart
disease .................................................................................................................... 43
Table (4.17): Relation of stent loci and in-stent restenosis ...................................... 43
X
List of figures
Figure (2.1): Coronary arteries of the heart. ............................................................... 4
Figure (2.2): Pathophysiology of coronary artery disease .......................................... 6
Figure (2.3): Cytogenetic location of CYP2C19 ........................................................ 8
Figure (2.4): Location of selected loss-of-function (*2-*8) and gain-of-function
(*17) variant alleles of CYP2C19 ............................................................................ 9
Figure (2.5): Chemical structure of clopidogrel ....................................................... 14
Figure (2.6): Pharmacokinetics of clopidogrel ......................................................... 15
Figure (2.7): Pharmacodynamic of clopidogrel ....................................................... 16
Figure (4.1): PCR products of CYP2C19*2&*3 ..................................................... 34
Figure (4.2): Restriction products for CYP2C19*2 by SmaI ................................... 35
Figure (4.3): Restriction products for CYP2C19*3 by BamH1 ............................... 36
Figure (4.4): CYP2C19 metabolizing status in the study population ....................... 39
Figure (4.5): distribution of patient according to restenosis ..................................... 39
Figure (4.6): Genotyping distribution of restenosis cases ........................................ 40
XI
List of Abbreviations
ADP The adenosine diphosphate
BMS Bare-Metal Stents
CABG Coronary Artery Bypass Graft
CAD Coronary Artery Disease
cAMP cyclic Adenosine Monophosphate
CAR Constitutive androstane receptor
cDNA Complementary DNA
COX-1 Acetylate Cyclooxygenase 1
CVD Cardiovascular Diseases
CYP Cytochrome P450
dbSNP The Single Nucleotide Polymorphism Database
DES Drug-Eluting Stents
EDTA Ethylenediaminetetraacetic acid
EGH European Gaza hospital
EM Extensive Drug Metabolizers
GATA4 Transcription factor GATA-4
GPIIb/IIIa Glycoprotein IIb/IIIa
GRα Human glucocorticoid receptor α
HNF3γ Hepatocyte nuclear factor 3-gamma
HNF4α Hepatic nuclear factors-4 alpha
IM Intermediate Metabolizers
LDL Low Density Lipoprotein
MARC Major adverse cardiac events
MOH Ministry Of Health –Palestine
PCI Percutaneous Coronary Intervention
PCR Polymerase Chain Reaction
PI3K Phosphatidylinositol 3-Kinase
PKB/Akt Serine-Threonine Protein Kinase B
PM Poor Metabolizers
PXR Pregnane X receptor
Rap1b Ras-related protein Rap-1b
RFLP Restriction Fragment Length Polymorphism
RPA Residual Platelet Aggregation
ST Stent Thrombosis
TAE Tris-acetate-EDTA
UM Ultrarapid Metabolizers
VASP Vasodilator-Stimulated Phosphoprotein
Chapter One
Introduction
1
Chapter 1
Introduction
1.1 Overview
Cardiovascular diseases (CVD) or heart diseases are a class of diseases that involve
the heart or blood vessels. Diseases under the heart disease umbrella include blood
vessel diseases, such as coronary artery disease (CAD), heart rhythm problems
(arrhythmias), heart infections and heart defects including congenital heart defects
(Gaziano, 2005; CDC, 2009 and Mozaffarian et al., 2015). Heart failure is a common
clinical syndrome that represents the final stage of a range of different heart diseases
(Anderson et al., 2007 and Ponikowski et al., 2014).
Coronary artery disease is a common form of CVD and it is the major source of
morbidity and mortality in the developing and developed countries (Walter, 2014). It
is responsible for an estimated 17.3 million deaths per year, a number that is
expected to grow to more than 23.6 million by 2030. (American heart association,
AHA, 2015). Coronary artery disease is caused by atherosclerosis which restricts
blood flow to the heart and when the blood flow is completely cut off, the result is
heart attack (CDC, 2009 and Sakakura, 2013). The pathogenesis of atherosclerosis
involves the processes of vascular injury, inflammation, degeneration and thrombosis
(Singh et al., 2006 and Weber and Noels, 2011). Percutaneous coronary intervention
(PCI) is a mainstay in the management of high-risk coronary artery disease and
requires anti-platelet therapy, depending on stent type and admission diagnosis (Zhou
et al., 2012 and Giustino et al., 2015).
Cytochrome P450 (CYP450) is the generic name given to a large family of enzymes
that metabolize most drugs and chemicals of toxicological importance. In humans,
there exist 18 mammalian CYP450 families, 44 subfamilies, and 57 putative
functional enzymes (Ingelman-Sundberg, 2005). The family members CYP1, CYP2,
and CYP3 of CYP450 represent the top candidate genes in pharmacokinetics (Yang
et al, 2010 and Knights et al., 2013). Four CYP2C genes have been identified in
humans: CYP2C8, CYP2C9, CYP2C18, and CYP2C19. CYP2C19 enzyme is one of
the hepatic cytochrome P450 enzymes that metabolizes many important clinical
2
drugs including antiulcer drug omeprazole, antiplatelet drug clopidogrel,
anticonvulsant mephenytoin bertilsson, antimalarial drug proguanilthe anxiolytic
drug diazepam, and certain antidepressants such as citalopram (Flachsbart et al.,
2011 and Lee, 2013).
There are 35 different variant alleles of CYP2C19 (OMIM, 2016). The CYP2C19*1
allele is the normal (wild-type) copy that has full enzymatic activity (Kubica et al.,
2011). The most common variant allele is named as CYP2C19*2 which result in
truncated, nonfunctional protein (Mega et al., 2009). Another common allele is
named as CYP2C19*3 which also creates a premature stop codon (Nakamoto et al.,
2007). Genetic polymorphisms of CYP2C19 are associated with impaired clopidogrel
metabolism in healthy and in patients (Sangkuhl et al., 2010).
Research on CYP2C19 enzyme among Gaza strip population is rare. Only one study
determined the frequencies of the major polymorphic CYP2C19 alleles (CYP2C19*2
and CYP2C19*3) and investigated association of their occurrence with childhood
hematological malignancies (Abu-Eid, 2006). No previous study linked CYP2C19
with the outcome of drug metabolism. The present study is the first to determine
cytochrome P450 2C19 polymorphism and to asses Clopidogrel management in
patients with CAD in Gaza Strip.
3
1.2 General objective
The general objective of the present study is to determine CYP2C19 polymorphism
and to asses clopidogrel management in patients with CAD in Gaza Strip.
1.3 Specific objectives
1. To determine the frequencies of the Cytochrome P450 2C19 wild type allele
(CYP2C19*1) and two polymorphic alleles (CYP2C19*2) and (CYP2C19*3)
in PCI patients.
2. To study the relevant clinical data of the PCI patients particularly data related
to their Clopidogrel treatment and any side effect or recurrence of
cardiovascular events.
3. To determine the importance of the loss of function alleles, if present, in
recurrence of cardiovascular events in PCI patients receiving Clopidogrel.
1.4 Significance
Cardiovascular disease is the first leading cause of death in Gaza strip and
PCI is commonly used in the management of CAD with anti-platelet therapy
clopidogrel.
CYP2C19 is responsible for Clopidogrel metabolic activation, and CYP2C19
loss-of function alleles appear to be associated with higher rates of recurrent
cardiovascular events in patients receiving clopidogrel.
The loss-of-function alleles are carried by different populations and
homozygotes, who are poor CYP2C19 metabolizers, make up 3% to 4% of
the population.
The present study is the first to assess the cytochrome P450 2C19
polymorphism and clopidogrel management in patients with CAD in Gaza
Strip, Palestine.
Chapter Two
Literature Review
4
Chapter 2
Literature Review
2.1 Heart and coronary arteries
The heart is a muscular (myocardium) organ that pumps blood throughout the blood
vessels to various parts of the body by repeated rhythmic contractions. The human
heart is located in the middle of the chest between the right and left lungs, and
slightly towards the left of the breastbone, anterior to the vertebral column and
posterior to the sternum and it is enclosed in a double-walled sac called the
pericardium (Starr et al., 2009). The heart has four chambers, two superior atria
(receiving chambers) and two inferior ventricles (discharging chambers).
Oxygenated blood returns from the lungs into the left atrium, which pumps it into the
left ventricle, whose subsequent strong contraction forces it out through the aorta
leading to the systemic circulation (Marieb, 2011). Coronary arteries arise from aorta
(Figure 2.1). The right coronary artery gives branches to the walls of the right atrium
and ventricle. The left coronary artery gives branches to supply the roots of the aorta
and pulmonary trunk and the walls of the left atrium and ventricle. The left coronary
artery bifurcates into the left anterior descending artery and the left circumflex artery.
Figure (2-1): Coronary arteries of the heart (Encyclopedia of science, 2016).
5
2.2 Definition of coronary artery disease
Coronary artery disease, also known as coronary heart disease (CHD), coronary
atherosclerosis and ischemic heart disease (IHD), which is a branch of CVD and a
common form of heart disease. Coronary artery disease is considered as an insidious
and dangerous disease in the world, and the major source of morbidity and mortality
in the developed world (Walter, 2014 and Rydén et al., 2013). It is caused by
atherosclerosis, an accumulation of fatty materials in the inner linings of arteries. The
resulting blockage restricts blood flow to the heart and when the blood flow is
completely cut off, the result is heart attack (CDC, 2009).
2.3 Mortality rate of cardiovascular diseases
According to World Health Organization fact sheet in 2015, CVD are the number
one cause of death globally, more people die annually from CVD than from any
other cause. An estimated 17.5 million people died from CVD in 2012, representing
31% of all global deaths. An estimated 7.4 million were due to CAD and 6.7 million
were due to stroke. Over three quarters of CVD deaths take place in low- and
middle-income countries. By 2030 about 23.6 million people will die from CVD,
mainly from CAD (Mozaffarian et al., 2015). Each year CVD causes over 2 million
deaths in the European Union (EU), representing 42% of all deaths in the EU (British
Heart Foundation, 2008). Cardiovascular disease accounted for 31% of all deaths in
Canada and CAD caused about half million deaths in USA (AHA, 2009). In the Arab
world, the proportion of death from CVD ranges from 17 to 21% (CDC, 2014). In
Palestine, CVD is the first leading cause of death. A total of 1088 cases from 3406 in
Gaza strip, with proportion of 31.9% died from CVD, and 1708 from 5581 with
proportion of 30.6% in the West Bank died from CVD (Ministry Of Health, 2010).
6
2.4 Pathophysiology of coronary artery disease
Coronary artery disease is caused by coronary artery atherosclerosis. The process of
arterial narrowing with atherosclerotic plaque development appears to be initiated by
endothelium injury (Hansson, 2005). Low density lipoprotein (LDL) compound
bounds to LDL receptor, internalized, and transported through the endothelium and
oxidative modification of LDL is happened in endothelium (Figure 2.2). The
macrophages remove the oxidized LDL via scavenger receptors by forming the foam
cells which are lipid filled macrophages (Rocha and Libby, 2009 and Walter, 2014).
The accumulated plaques of core lipid, thick fibrous filled with inflammatory cells as
macrophage and T cells may be ruptured in response to the physical forces of blood
flowing inside swelling of artery wall (Stocker et al., 2004). Blood platelets begin to
accumulate at the site of a vulnerable coronary plaque and to initiate thrombotic
occlusion of the coronary vessel (Gawaz, 2004 and Rocha and Libby, 2009).
Figure (2.2): Pathophysiology of coronary artery disease (Rocha and Libby, 2009).
LDL= Low density lipoprotein
7
2.5 Risk factor of coronary artery disease
The common risk factors of CAD include socioeconomic status (Cunningham,
2010), family history of the disease (Hasanaj et al. 2013 and Kulkarni 2015),
smoking (Sadeghi et al., 2013), physical activity (Stewart et al., 2013), obesity
(Lavie et al., 2009 and Abed and Jamee 2015), hypertension and diabetes (Chiha et
al., 2012 and Bhalli et al., 2015).
2.6 Cytochrome P450
2.6.1 Definition
The cytochrome P450 superfamily (also called CYP) is the generic name given to a
large family of heme-containing enzymes embedded primarily in the lipid bilayer of
the endoplasmic reticulum of hepatocytes (Bibi, 2008).
2.6.2 Nomenclature of CYP
A nomenclature system for CYP enzymes is based on the degree of similarity of
primary amino acid sequences to other CYPs, if there it have a higher amino acid
sequence homology than 40% they grouped into families (e.g. CYP1 vs. CYP2), and
those with greater than 55% homology are grouped into subfamilies (e.g. CYP2C)
(Lamb and Waterman, 2013). The root symbol CYP for cytochrome P450, an arabic
number for the CYP450 family, a letter for the subfamily, an arabic numeral for the
individual gene (e.g. CYP2C19) and when describing a CYP450 gene, all letters and
numerals are written in italics (McKinnon, 2008).
2.6.3 Classification of CYP
In humans, the enzymes responsible for drug metabolism belong to the CYP families
are CYP1, CYP2, CYP3 and CYP4. The CYP2 is the largest family of cytochrome
and includes 13 subfamilies that consist of 16 functional genes and 13 confirmed
pseudogenes. The CYP2C is the largest subfamily of CYP2 enzymes located at the
chromosomal position 10q24. Four members of this subfamily have been identified,
namely CYP2C8, CYP2C9, CYP2C18, and CYP2C19. All members of this subfamily
exhibit genetic polymorphisms, particularly, the CYP2C9 and CYP2C19 (Goldstein,
2001; Nakamoto et al., 2007 and Rainone et al., 2015).
8
2.6.4 Function of CYP
CYP enzymes are responsible for 75-80% of all phase I-dependent metabolism and
for 65-70% of the clearance of clinically used drugs (Sim and Ingelman-sundberg,
2010). The enzyme CYP2C19 functions in the metabolism of many drugs, including
antiplatelet, antidepressants and proton pump inhibitors (Li-Wan-Po et al., 2010).
2.7 Cytochrome P450 2C19
2.7.1 Definition and function
The CYP2C19 enzyme is defined as homo sapiens cytochrome P450, family 2,
subfamily C, polypeptide 19 (CYP2C19) (NCBI, 2015). The CYP2C19 enzyme is a
protein of 490 amino acids with molecular mass of 55931 Da, found primarily in the
liver (The GeneCards human gene database, 2015). The most commonly known
substrates for CYP2C19 include proton pump inhibitors (PPIs), certain
antidepressants (citalopram/escitalopram, imipramine), antiepileptics (diazepam,
mephenytoin), the antimalarial drug proguanil, the β-adrenoceptor blocker
propranolol and the antiplatelet drug clopidogrel (Chang M, 2014).
2.7.2 CYP2C19 gene
The CYP2C19 gene is located on the long (q) arm of chromosome 10 at position 24
(10q24.1–q24.3) and it has 9 coding exons and 8 introns (Figure 2.3). More
precisely, the CYP2C19 gene is located from base pair 94,762,680 to base pair
94,853,204 on chromosome 10, the cDNA is 1473-bp in length (Trenk et al., 2008
and Genetics Home Reference, 2015).
Figure (2.3): Cytogenetic location of CYP2C19 (Genetics Home Reference, 2015).
9
2.7.3 Expression of CYP2C19
The CYP2C19 is predominantly expressed in the liver and, to a lesser extent, in the
small intestine. Expression of CYP2C19 is largely mediated by hepatic nuclear
factors-4 alpha (HNF4α) and Hepatocyte nuclear factor 3-gamma (HNF3γ).
Transcriptional activation is mediated by the drug-responsive nuclear receptors
of constitutive androstane receptor (CAR), the pregnane X receptor (PXR), and the
human glucocorticoid receptor α (GRα). In-vitro expression studies have recently
shown that the transcription factor GATA-4 (GATA4) also up regulates CYP2C19
transcriptional activity (Scott et al., 2012).
2.7.4 Polymorphism of CYP2C19
To date, 35 CYP2C19 variants have been identified (OMIM, 2016). The CYP2C19*1
normal allele is associated with functional CYP2C19-mediated metabolism (Scott et
al. 2013). Location of selected loss, reduced and gain-of-function alleles of
CYP2C19 are shown in Figure 2.4 and Table 2.1 (Scott et al., 2012).
Figure (2.4): Location of selected loss-of-function (*2–*8) and gain-of-function
(*17) variant alleles of CYP2C19. Exons are represented by numbered black boxes.
(Scott et al., 2012).
10
2.7.4.1 The CYP2C19*2
The most common loss-of-function allele is CYP2C19*2 (previously referred to as
CYP2C19m1) with frequencies of 15% in Caucasians and Africans, and 29–35% in
Asians (Table 2.1). The CYP2C19*2 result from point mutation in exon 5, with
a G-A nucleotide change at 681 of cDNA sequence (defined by a 681G>A
substitution, rs4244285) (Mega et al., 2009). This activates a new cryptic site 40
nucleotides downstream of the natural splice site. This change alters the mRNA
reading frame creating truncated, nonfunctional protein (Rogan et al., 2003).
CYP2C19*2 is inherited as an autosomal co-dominant trait (Dean L, 2013). Sub-
alleles of CYP2C19*2 have been identified that harbor additional SNPs with limited
or no added functional consequence (i.e. CYP2C19*2A, *2B, *2C, *2E, *2F, *2G,
*2H, and *2J) (Sim, 2010). Platelet responsiveness to clopidogrel in heterozygotes
(*1/*2) lies somewhere between the responsiveness in individuals with the *1/*1
genotype and that in those with the *2/*2 genotype (Scott et al., 2013).
2.7.4.2 The CYP2C19*3
Another common allele is CYP2C19*3 (previously referred to as CYP2C19m2)
(Table 2.1) which has a G-A mutation at position 636 of exon 4 that result in a
premature termination codon at amino acid 212 which also creates a Stop codon
gained (defined by a 636G>A substitution, rs4986893) producing a truncated
inactive enzyme (Danielson, 2002 and Nakamoto, 2007). Sub-alleles of CYP2C19*3
also identified that harbor additional SNPs with limited or no added functional
consequence (CYP2C19*3, *3A, *3B and *3C) (Sim, 2010). The CYP2C19*3, is rare
among Caucasian subjects but accounts with frequencies typically <1%, with the
exception of CYP2C19*3 in Asians (2-9%) (Scott et al., 2011).
2.7.4.3 Other CYP2C19 variants that encode reduced or unknown
enzymatic activity
Less frequent CYP2C19 alleles associated with absent or reduced enzyme activity
are CYP2C19*4 (rs28399504), *5 (rs56337013), *6 (rs72552267), *7 (rs72558186),
and *8 (rs41291556) (Table 2.1). These variants typically have allele frequencies less
than 1% (Scott et al., 2012).
11
Table (2.1): Commonly CYP2C19 variant alleles and their effect on CYP2C19
protein.
Allele Nucleotide changes dbSNP Enzyme activity
cDNA Gene
CYP2C19*1 Normal
CYP2C19*2 681G>A 19154G>A rs4244285 None
CYP2C19*3 636G>A 17948G>A rs4986893 None
CYP2C19*4 1A>G 1A>G rs28399504 None
CYP2C19*5 1297C>T 90033C>T rs56337013 None
CYP2C19*6 395G>A 19294T>A rs72552267 None
CYP2C19*7 12748G>A rs72558186 None
CYP2C19*8 358T>C 12711T>C rs41291556 Reduced
CYP2C19*17 –806C>T rs12248560 Increased
(Scott et al., 2012).
2.7.4.4 CYP2C19 Variants that encode increased enzymatic activity
A novel allelic variant of cytochrome P450 2C19 encoding ultrarapid enzyme
activity was described (denoted CYP2C19*17) (Rudberg et a., 2008). The
CYP2C19*17 allele is associated with increased enzymic activity. However, the
magnitude of effects is considerably smaller than has been reported with CYP2C19*2
and CYP2C19*3, albeit in opposite directions (Li-Wan-Po et al., 2010). The
rs12248560 (c. −806C>T) is the defining polymorphism of the CYP2C19*17 allele
and is a C>T transition in the promoter that creates a consensus binding site for the
GATA transcription factor family, resulting in increased CYP2C19 expression and
activity (Table 2.1). The CYP2C19*17 allele frequencies are approximately 21% in
Caucasians, 16% in African-Americans, and 3% in Asians (Scott et al., 2012).
2.7.5 Impact of CYP2C19 (polymorphism) genotype on drug metabolism
Based on the ability to metabolize CYP2C19 substrates, individuals can be
categorized as extensive drug metabolizers (EM), where they are homozygous for
the CYP2C19*1 allele, which is associated with functional CYP2C19-mediated
metabolism (*1/*1), intermediate metabolizers (IM) which contains one wild-type
12
allele and one variant allele that encodes reduced or absent enzyme function (e.g.,
*1/*2, *1/*3), resulting in decreased CYP2C19 activity, poor metabolizers (PM)
have two loss-of-function alleles (e.g., *2/*2, *2/*3, *3/*3), resulting in markedly
reduced or absent CYP2C19 activity and ultrarapid metabolizers (UM) (*1/*17,
17*/*17) (Desta et al., 2002). There are marked racial differences in the frequencies
of CYP2C19 phenotypes, with Asians having the highest frequencies of the poor
metabolizers and intermediate metabolizer phenotypes. Some studies showed that *2
and *3 could explain more than 90% poor metabolizers phenotypes but other studies
indicate that *2 and *3 could explain less than 50% of poor metabolizers (Nakamoto
et al., 2007 and Cavallari et al., 2011).
2.8 Management of coronary artery disease
The key components in the management of CAD include coronary revascularization,
antiplatelet therapy, anticoagulation and consideration of adjuvant agents including β
blockers, inhibitors of the renin angiotensin system, and 3-hydroxy-3-methyl-
glutaryl-CoA reductase inhibitors (Smith et al., 2015).
2.8.1 Coronary revascularization
2.8.1.1 Balloon angioplasty
Balloon angioplasty (called cardiac catheterization) is an invasive, non-surgical
procedure done under local anesthesia to study the coronary arteries. During the
procedure, a small hollow tube (catheter) that has a small balloon on its tip is inserted
into an artery of the wrist or groin. They inflate the balloon at the blockage site in the
artery to flatten or compress the plaque against the artery wall. Special X-ray dye or
contrast is injected through the catheter into the arteries. This will outline the
coronary arteries to show any existing blockages or narrowing. Most patients have
minimal discomfort during cardiac catheterization (American Heart Association.
2010).
13
2.8.1.2 Percutaneous coronary intervention (PCI)
Percutaneous coronary intervention is a mainstay in the management of high-risk
coronary artery disease. Initially the patient has a cardiac catheterization to
visualize the position and shape of any narrowing or blockages. If the clinical
circumstances and the cardiac catheterization findings suggest blood flow to the
heart must be modified, the majority of patients will be treated by PCI and minority
will be treated by coronary artery bypass graft (CABG) (Redwood, 2011).
During PCI, a small, hollow metal mesh tube called a stent is placed in the artery to
keep it open following initial balloon dilation. Stents are classified into drug-eluting
stents (DES) and bare-metal stents (BMS). Drug-eluting stents are used in the
majority of patients who receiving intracoronary stents (Balghith et al., 2013). In
patients with CAD, randomized controlled trials have consistently linked DES with
reduction of neointimal hyperplasia, decreased risk of restenosis compared with
BMS (Jensen et al., 2007). Drug-eluting stents decreases the frequency of repeat
revascularization procedures in patients with CAD undergoing PCI and at highest
risk for restenosis, reducing rate of death or myocardial infarction (Tu et al., 2007
and Capodanno et al., 2011). However, stenting itself requires potent peri-
procedural antithrombotic therapy and a commitment to ≥ 1–6 months of
antiplatelet therapy, depending on stent type and admission diagnosis (Zhou et al.,
2012).
2.8.2 Antiplatelet therapy
Platelets play an important role in cardiovascular disease both in the pathogenesis of
atherosclerosis and in the development of acute thrombotic events. Their importance
in vascular disease is indirectly confirmed by the benefit of antiplatelet agents in
these disorders (Norgard and Abu-Fadel, 2009). Anti-platelet medications are the
cornerstone of treatment for patients with cardiovascular disease and undergoing
percutaneous coronary intervention (PCI). (Perry and Shuldiner, 2013). For many
years, aspirin has been the mainstay of antiplatelet drug therapy in vascular disease.
As an antiplatelet agent, aspirin has been shown to greatly reduce major vascular
adverse events. Its benefit it has been linked to its ability to permanently acetylate
cyclooxygenase 1 (COX-1), preventing the conversion of arachidonic acid to
14
thromboxane A2 by the platelet. Thromboxane A2 is a strong platelet agonist and
inhibiting its production decreases overall platelet aggregation at the site of the
vascular injury. However, blocking this pathway has a limited overall effect on the
various independent pathways (i.e., collagen and adenosine diphosphate) can remain
high. The need for inhibition of other platelet activation pathways has led to the
development of additional antiplatelet drugs (Norgard and Abu-Fadel, 2009).
2.8.2.1 P2Y12 receptor antagonists
The adenosine diphosphate (ADP) receptor inhibitors are a subclass of anti-platelet
medications which include clopidogrel, prasugrel, ticagrel and ticlopidine (perry and
shuldiner, 2013). ADP is an essential agonist in hemostasis and thrombosis that
mediates its effects through the P2Y1 and P2Y12 platelet receptors. Ticlopidine and
Clopidogrel are commonly used thienopyridine anti-platelet agents that selectively
and irreversibly block the P2Y12 ADP receptor. Clopidogrel is favored over
Ticlopidine due to better patient safety and tolerability (Norgard and Abu-Fadel,
2008).
2.9 Clopidogrel
2.9.1 Definition and structure
Clopidogrel is an oral thienopyridine derivative which inhibits platelet activation
and aggregation by irreversibly blocking the platelet adenosine diphosphate (ADP)
P2Y12 receptor (Ma et al., 2011). Chemically clopidogrel is (d-methyl[2-
chlorophenyl]-5-[4,5,6,7-tetrahydrothieno] [3,2-c pyridinyl] acetate hydrogensulfate)
having the empirical formula C16H17ClNO2S.HSO4 and molecular mass
321.82 g/mol, (Figure 2.5, Gurbel and Tantry, 2007).
Figure (2.5): Chemical structure of clopidogrel (Gurbel and Tantry, 2007).
15
2.9.2 Pharmacokinetics of clopidogrel
Clopidogrel is a pro-drug that is absorbed in the intestine and activated in the liver
(Figure 2.6). The P-glycoprotein is responsible for the active intestinal transport of
clopidogrel (Giusti et al., 2010). Clopidogrel is metabolized by two main metabolic
pathways: an esterase-dependent pathway leading to hydrolysis up to 85 % of
clopidogrel into an inactive carboxylic acid derivative and a cytochrome P450
(CYP)-dependent pathway (Karaźniewicz-Łada et al., 2014) leading to formation of
its active metabolite clopi-H4. Clopi-H4 is formed in a two-step oxidative process
mediated by CYP1A2, CYP2B6, CYP2C19 and CYP3A4 (Dansette et al., 2011),
which bind to the ADP, P2Y12 receptor expressed on the platelet surface, and causes
an irreversible blockade of ADP-binding for the platelet’s life span (Ma et al., 2011).
Figure (2.6): Pharmacokinetics of clopidogrel (Giusti et al., 2010).
2.9.3 Pharmacodynamic of clopidogrel
Clopidogrel non-competitively and irreversibly inhibits the adenosine diphosphate
(ADP) P2Y12 receptor; ADP binds to the P2Y1 receptor to induce change in platelet
shape and to initiate a weak and transient phase of platelet aggregation (Angiolillo et
al., 2007 and Angiolillo and Ferreiro, 2010). The binding of ADP to its Gi-coupled
P2Y12 receptor liberates the Gi protein subunits i and . The subunit i leads to the
16
inhibition of adenylyl cyclase, which, in turn, lowers the cyclic adenosine
monophosphate (cAMP) level. This inhibits the cAMP-mediated phosphorylation of
vasodilator-stimulated phosphoprotein (VASP) (VASP-P), which inhibits glyprotein
IIb/IIIa receptor activation. The subunit activates the phosphatidylinositol 3-
kinase (PI3K), which leads to GP IIb/IIIa receptor activation through activation of a
serine-threonine protein kinase B (PKB/Akt) and of Rap1b GTP binding proteins.
The Gi-coupled P2Y12 receptor pathway results in stabilization of platelet
aggregation. Thus, blockage of P2Y12 receptor effectively inhibits such platelet
aggregation (Figure 2.7).
Figure (2.7): Pharmacodynamic of clopidogrel
AC= Adenylyl Cyclase, cAMP= cyclic Adenosine Monophosphate, VASP=
Vasodilator-Stimulated Phosphoprotein, PI3K= Phosphatidylinositol 3-Kinase,
GPIIb/IIIa= Glycoprotein IIb/IIIa, PKB/Akt= Serine-threonine Protein Kinase B,
Rap1b: Ras-related protein Rap-1b (Angiolillo and Ferreiro, 2010).
17
2.10 Prevalence and relation of CYP2C19*2 and *3 alleles to the
platelet inhibition by clopidogrel
Hulot et al., 2006 conducted a prospective pharmacogenetic study in 28 healthy
French white male volunteers treated for 7 days with clopidogrel 75 mg/day to
determine whether frequent functional variants of genes coding for candidate CYP
isoenzymes involved in clopidogrel metabolic activation (CYP2C19*2, CYP2B6*5,
CYP1A2*1F, and CYP3A5*3 variants) influence the platelet responsiveness to
clopidogrel. Results showed that pharmacodynamic response to clopidogrel was
significantly associated with the CYP2C19 genotype. Twenty of the subjects were
wild-type CYP2C19 (*1/*1) homozygotes, while the other 8 subjects were
heterozygous for the loss-of-function polymorphism CYP2C19*2 (*1/*2). Baseline
platelet activity was not influenced by the CYP2C19 genotype. In contrast, platelet
aggregation in the presence of 10 µM ADP decreased gradually during treatment
with clopidogrel 75 mg once daily in *1/*1 subjects, reaching 48.9%±14.9% on day
7 (P<0.001 vs baseline), whereas it did not change in *1/*2 subjects (71.8%±14.6%
on day 7, P=0.22 vs baseline, and P<0.003 vs *1/*1 subjects). Similar results were
found with vasodilator-stimulated phosphoprotein phosphorylation. The CYP2C19*2
loss-of-function allele is associated with a marked decrease in platelet responsiveness
to clopidogrel in young healthy male volunteers and may therefore be an important
genetic contributor to clopidogrel resistance in the clinical setting.
The relationship between genetic variation in CYP isoenzymes and the
pharmacokinetic/pharmacodynamic response to prasugrel (60 mg, n=71) and
clopidogrel (300 mg, n=74) was determined in healthy subjects from Netherlands
(Brandt et al., 2007). Genotyping was performed for CYP1A2, CYP2B6, CYP2C19,
CYP2C9, CYP3A4 and CYP3A5. In subjects receiving clopidogrel, the presence of
the CYP2C19*2 loss of function variant was significantly associated with lower
exposure to clopidogrel active metabolite, as measured by the area under the
concentration curve (AUC(0-24); P=0.004) and maximal plasma concentration
(C(max); P=0.020), lower inhibition of platelet aggregation at 4 hr (P=0.003) and
poor-responder status (P=0.030). Similarly, CYP2C9 loss of function variants were
significantly associated with lower AUC(0-24) (P=0.043), lower C(max) (P=0.006),
18
lower IPA (P=0.046) and poor-responder status (P=0.024). For prasugrel, there was
no relationship observed between CYP2C19 or CYP2C9 loss of function genotypes
and exposure to the active metabolite of prasugrel or pharmacodynamic response.
Trenk et al. (2008) investigated whether the loss of function CYP2C19*2
polymorphism is associated with high (>14%) residual platelet aggregation (RPA) on
clopidogrel and whether high on-clopidogrel RPA impacts clinical outcome after
elective coronary stent placement. The study included 797 consecutive Germany
patients undergoing PCI, who were followed-up for one year. Adenosine-
diphosphate-induced (5 mumol/l) RPA was assessed after a 600-mg loading dose and
after the first 75-mg maintenance dose of clopidogrel before discharge. CYP2C19
genotype was analyzed by real-time PCR. Of the patients included, 552 (69.3%)
were CYP2C19 wild-type homozygotes (*1/*1) and 245 (30.7%) carried at least one
*2 allele. Residual platelet aggregation at baseline did not differ significantly
between genotypes. On clopidogrel, RPA was significantly (P<0.001) higher in *2
carriers than in wild-type homozygotes (23.0% [interquartile range (IQR) 8.0% to
38.0%] vs. 11.0% [IQR 3.0% to 28.0%] after loading; 11.0% [IQR 5.0% to 22.0%]
vs. 7.0% [IQR 3.0% to 14.0%] at pre-discharge). Between *2 carriers and wild-type
homozygotes, significant (P<0.001) differences in the proportion of patients with
RPA >14% was found, both after loading (62.4% vs. 43.4%) and at pre-discharge
(41.3% vs. 22.5%). Residual platelet aggregation >14% at pre-discharge incurred a
3.0-fold increase (95% confidence interval 1.4 to 6.8; p = 0.004) in the one-year
incidence of death and myocardial infarction.
In their study entitled “Cytochrome P-450 polymorphisms and response to
clopidogrel”, Mega et al (2009) found that 30% of the study population, carriers of at
least one CYP2C19 reduced-function allele, had a relative reduction of 32.4% in
plasma exposure to the active metabolite of clopidogrel, as compared with
noncarriers (P<0.001). Carriers also had an absolute reduction in maximal platelet
aggregation in response to clopidogrel that was 9% points less than that seen in
noncarriers (P<0.001). Among clopidogrel-treated subjects in Therapeutic Outcomes
by Optimizing Platelet Inhibition with Prasugrel–Thrombolysis in Myocardial
19
Infarction, carriers had a relative increase of 53% in the composite primary efficacy
outcome of the risk of death from cardiovascular causes, myocardial infarction, or
stroke, as compared with noncarriers (12.1% vs. 8.0%; hazard ratio for carriers, 1.53;
95% confidence interval [CI], 1.07 to 2.19; P=0.01) and an increase by a factor of 3
in the risk of stent thrombosis (2.6% vs. 0.8%; hazard ratio, 3.09; 95% CI, 1.19 to
8.00; P=0.02).
Buzoianu et al. (2010) studied CYP2C19*2, *3 and *4 variants in 200 healthy,
unrelated Romanian volunteers, using Polymerase Chain Reaction- restriction
Fragment Lengh Polymorphism (PCR-RFLP) and tetra-primer PCR techniques.
Forty eight individuals (24%) were CYP2C19*2 heterozygotes, while a homozygous
genotype CYP2C19*2/*2, has been demonstrated in 3 individuals (1.5%). No
CYP2C19*3 variant has been found. CYP2C19*4 was found only in a
CYP2C19*2/*4 compound heterozygous individual. The allele frequencies for
CYP2C19*2, *3 and *4 were 13.75%, 0% and 0.25%, respectively.
The CYP2C19*1, *2 and *3 variants were assessed by PCR-RFLP assay in a
representative sample of 161 unrelated healthy Lebanese volunteers (Jureidini et al.,
2011). The allele frequencies of CYP2C19*2 and *3 were 0.13 and 0.03. Carriers of
the CYP2C19*2 or *3 represented 24.2% of the subjects. In addition, Ellison et al.
(2012) found that the frequency of the CYP2C19 genotype combinations were
CYP2C19 *1/*1 (93%), *1/*2 (6%), and *2/*2 (1%) in Egyptian population. The
observed allele frequency of CYP2C19 681G>A was found 3.8%.
Yousef et al. (2012) determined the frequencies of important allelic variants of
CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 in the Jordanian population and
compare them with the frequency in other ethnic groups. Genotyping of CYP1A1(m1
and m2), CYP2C9 (2 and 3), CYP2C19 (2 and 3), CYP3A4 5, CYP3A5 (3 and 6), was
carried out on Jordanian subjects. Different variants allele were determined using
PCR-RFLP. CYP1A1 allele frequencies in 290 subjects were 0.764 for CYP1A1 1,
0.165 for CYP1A1 2A and 0.071 for CYP1A1 2C. CYP2C9 allele frequencies in 263
subjects were 0.797 for CYP2C9*1, 0.135 for CYP2C9*2 and 0.068 for CYP2C9*3.
20
For CYP2C19, the frequencies of the wild type (CYP2C19*1) and the nonfunctional
*2 and *3 alleles were 0.877, 0.123 and 0, respectively. Five subjects (3.16 %) were
homozygous for 2/2. Regarding CYP3A4 1B, only 12 subjects out of 173 subjects
(6.9 %) were heterozygote with none were mutant for this polymorphism. With
respect to CYP3A5, 229 were analyzed, frequencies of CYP3A5 1, 3 and 6 were
0.071, 0.925 and 0.0022, respectively. Comparing our data with that obtained in
several Caucasian, African-American and Asian populations, Jordanians are most
similar to Caucasians with regard to allelic frequencies of the tested variants of
CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5.
Abid et al. (2013) investigated the genetic variant of the gene CYP 2C19 in 100
Tunisian patients and assessed the involvement of this genetic profile in the
occurrence of major cardiovascular events (MACE) during the follow-up period. The
patients were divided into 2 groups: those with at least one CYP2C19*2 allele (*2
carriers) and non-carriers. The mean age of patients was 56.7±10.5 years. The
prevalence of CYP2C19*2 allele was 11.5%. Hospital mortality was estimated at
3%. No statistically significant differences were noted between the two groups
regarding the occurrence of intra hospital MACE. The mean follow up was 7.5 ±
4.87 months for the entire study population. The rate of MACE during the follow-up
of patients receiving clopidogrel was 8.2% throughout the study population: 5.3% in
the *2 non-carriers versus 18.2% in the *2 carriers with a statistically significant
difference (P=0.075) at the risk of error of 10%. Concerning the occurrence of stent
thrombosis, there was no significant statistical difference between the two study
groups.
The prevalence of CYP2C19*2 and ABCB1 C3435T polymorphisms in 100
unrelated Palestinian subjects and 100 unrelated Turkish subjects was examined by
the amplification refractory mutation system (Nassar et al., 2014). Results showed an
ABCB1 3435 T allele frequency of 0.46 (95% CI 0.391 to 0.529) in the Palestinian
sample and 0.535 (95% CI 0.4664 to 0.6036) in the Turkish sample. CYP2C19*2
allele frequency was 0.095 (95% CI 0.0558 to 0.134) in the Palestinian sample and
0.135 (95% CI 0.088 to 0.182) in the Turkish sample.
Chapter Three
Material and Methods
21
Chapter 3
Material and Methods
3.1. Study design
A cross sectional study with convenience sample.
3.2. Study population
The study population involved all patients attending the European Gaza Hospital
(EGH), Cardiac Catheterization Department for follow up after percutaneous
coronary intervention and are being managed with the antithrombotic drug
Clopidogrel. The samples were collected during the period from the beginning of
March to the end of May 2014.
3.3. Sample size
A total of 110 patients were recruited from the Cardiology Department of EGH. The
sample covered the five main Governorates of Gaza strip: North Gaza, Gaza city,
Mid-Zone, Khan Younis and Rafah. The sample consisted of 73 males and 37
females with ages ranging from 34 to 88 years old with mean age of 60.2 ± 11.2
years.
3.4. Ethical consideration
The study was approved by the Palestinian Ethical Committee (Helsinki Ethics
Committee). (Appendix 1). Also, the approval of the Ministry of Health (MOH)
(Appendix 2) was obtained. Patient's consents were obtained orally to undergo this
study and collecting blood samples for analysis, after explaining the aim and
objectives of the study.
3.5. Inclusion criteria
Patients with the following 3 criteria were recruited in the study
Acute coronary syndrome patients who underwent PCI
Unrelated men and women patients
Patients treated with clopidogrel and aspirin
22
3.6. Exclusion criteria
Patients untreated with clopidogrel
3.7. Materials
3.7.1. Instruments
Table 3.1 list of instruments used to carry out the experimental work. All instruments
are located at the laboratories of the biotechnology department of the Islamic
University of Gaza.
Table (3.1): List of laboratory instruments
Item Manufacturer
Thermocycler Biometra, Germany
Electrophoresis chambers and tanks
(horizontal) BioRad, USA
Electrophoresis power supply BioRad, USA
Microcentrifuge BioRad, USA
Microwave oven LG, Korea
Freezer, refrigerator ORSO, pharml-spain
Safety cabinet Hereaus, Germany
Micropipettes (0.1-2.5μl / 0.5-10μl / 5-50μl /
20-200μl / 100-1000μl) Eppendrof, Germany
Gel documentation system Vision, Scie-Plas Ltd, UK
Digital camera Canon (Japan)
Vortex mixer Labnet, USA
Nano-drop spectrophotometer Implen, Germany
23
3.7.2. List of reagents and chemicals (Table 3-2)
Table (3.2): Lists the chemicals and reagents
Reagents and chemicals Manufacturer
Primers Hy.Labs®, (Rehovot, Israel)
Wizard Genomic DNA purification kit Promega, (Madison, USA)
Ladder (size marker) 100bp GeneDire X, USA
GoTaq® Green Master Mix Promega, (Madison, USA)
SmaI and BamHI Restriction enzymes New England BioLab, Canada
Agarose gel Promega, (Madison, USA)
Ethedium promide Promega, (Madison, USA)
Ultra-pure water (nuclease free) Promega, (Madison, USA)
Tris Acetate EDTA (TAE) (50X) Promega, (Madison, USA)
Ethanol >96% (Sigma USA).
Absolute Isopropanol (Sigma USA).
3.7.3. Primers used in this study (Table 3.3)
Table (3.3): Nucleotide sequence of the PCR primers
Primers Reference
ID Sequence (5, to 3
,)
CYP2C19*2-F CAGAGCTTGGCATATTGTATC
Yang et al., 2004
CYP2C19*2-R GTAAACACACAACTAGTCAATG
CYP2C19*3-F AAATTGTTTCCAATCATTTAGCT
CYP2C19*3-R ACTTCAGGGCTTGGTCAATA
24
3.7.4. Disposables (Table 3-4)
Table (3.4): List of disposables
Item Manufacturer
Steril 1.5ml eppendrof safe-lock
microcentrifuge tube Eppendrof
®, USA
Sterile thin-wall polypropylene PCR
tubes with attached caps for labeling Labcon USA
Disposable tips (different sizes from
0.2µl to 1000µl) Different manufacturers
2.5 K3 EDTA tubes ZMC, China
Syringe 5ml Homed – China
23 gauge needle Homed – China
3.8. Methods
3.8.1. Data collection
Data were recorded on a predesigned form (Appendix 3) after explaining the aim of
the study and obtaining the patient consent to participate in the study and donate a
blood sample. Personal data were verbally collected from the patient (name, age,
address, education level, family history of heart disease). Current and previous
clinical and laboratory data were collected from the medical record of the patient.
Similarly, the collected data included the medications and doses administered since
the PCI procedure. The patients were contacted by telephone to follow up for the
patient general condition and for any cardiovascular problems or complications that
might occurred during the last 6 months following PCI
3.8.2. Blood samples collection
Whole blood samples were collected from 110 patients in EDTA tubes.
Approximately 2.5 ml venous blood samples were collected in each tube from each
patient.
25
3.9. Molecular analysis
3.9.1. Extraction and purification of genomic DNA
DNA was extracted from blood samples using the Wizard Genomic DNA
purification Kit (Promega, Madison, WI. USA), according to the manufacturer
instructions protocol. The kit contents are listed in Table 3.5, and materials that
should be supplied by the user are indicated in Table 3.6. The extraction procedure is
as follows:
Cell Lysis
A volume of 300µl well mixed blood was added into a 1.5 ml microcentrifuge
tube containing 900 µl of the cell lysis solution (lyses of red blood cells). The
components were mixed by inverting the tube gently and the mixture was
incubated at room temperature for 10 minutes. During the incubation period, the
tube was periodically mixed (2-3 times) by inversion.
The mixture was centrifuged at 13,000 rpm for 20 seconds, then the supernatant
was removed and discarded without disrupting the pellet. The pellet was then
resuspended by vigorous vortexing ( for 10-15 seconds).
Nuclei lysis and protein precipitation
A volume of 300 µl nuclei lysis solution (lyses of white blood cells and their
nuclei) were added to the resuspend pellet and mixed by 5- 6 times pipetting to
obtain viscous cell extract.
One hundred µl protein precipitation solution (precipitates all the cellular and
nuclear proteins) were added to the lysate and vigorously mixed by vortex. The
extract was then centrifuged at 13,000 rpm for 3 minutes to precipitate the dark
brown protein pellet.
DNA precipitation and rehydration
The supernatant was then transferred to a clean 1.5 ml microcentrifuge tube
containing 300 µl isoprobanol and was mixed gently by inversion until white
thread-like strands of DNA formed a visible mass, then centrifuged at 13000 rpm
for 1 minute to pellet the DNA.
26
The supernatant was removed and 300µl of 70% ethanol were added and gently
inverted several times to wash the DNA pellet, then centrifuged at 13,000 rpm for
1 minute.
The ethanol was aspirated carefully using a micropipette, and then the tube was
inverted on a clean absorbent paper.
The pellet was air-dried for 10-15 minutes. The DNA pellet was rehydrated by
addition of 100µl DNA rehydration solution and incubation at 65 oC for 1 hour.
Finally The DNA was stored at -20 oC until performing PCR.
Table (3.5): Wizard genomic DNA purification kit solutions
Solutions Volume
Cell lysis solution 100 ml
Nuclei lysis solution 50 ml
Protein precipitation solution 25 ml
DNA rehydration solution 50 ml
RNase solution (4mg/ml) 250 µl
Table (3.6): Materials that should be supplied by the user
Sterile 1.5 ml microcentrifuge tubes
Water bath
Isopropanol, room temperature.
70% ethanol, room temperature.
Microcentrifuge.
27
3.9.2. Primers reconstitution
The primers were reconstituted to obtain 100 pmol/µl stock concentration from
which working primer dilutions were prepared at 5 pmol/µl concentration by
combining 5 µL of each reconstitute and 95 µL of ultra-pure water.
3.10. Detection of CYP2C19*2 and CYP2C19*3 polymorphisms by
PCR-RFLP
The procedure were performed according to previously published protocols (Yang et
al ., 2004), using the forward primer (CYP2C19*2-F), which anneals in intron 4 (71
bp upstream from the intron 4/exon 5 junction), and the reverse primer (CYP2C19*2-
R), which anneals in intron 5 (73 bp downstream from the exon 5/intron 5 junction).
After PCR amplification, the DNA fragments of exon 5 were digested with SmaI
before electrophoresis in 2.5% agarose gel for detection of the CYP2C19*2
fragments. For the identification of CYP2C19*3, the forward primer (CYP2C19*3-F)
anneals in intron 3 (21 bp upstream of the intron 3/exon 4 junction), and the reverse
primer (CYP2C19*3-R) in intron 4 (88 bp downstream of the exon 4/intron 4
junction). The PCR products for detecting CYP2C19*3 were digested with BamHI
and analyzed using gel electrophoresis.
3.10.1. Polymerase Chain Reaction (PCR) for CYP2C19*2
PCR for the CYP2C19*2 polymorphism, was carried out in a 25 µl reactions. The
reaction components were combined in 0.2 ml PCR tubes as described in (Table 3-7).
The tubes were then placed in a thermocycler and PCR amplification was performed
according to the program provided in (Table 3-8).
28
Table (3.7): PCR components for amplification of CYP2C19*2
Reagent Volume (μl) Final concentration
GoTaq® Green Master Mix (2X) 12.5 1X
CYP2C19*2-F (5 pmol/µl) 1 0.2 pmol/µl
CYP2C19*2-R 1 0.2 pmol/µl
DNA (25- 40 ng/µl) 3 About 100 ng in total
Nuclease free water 7.5
Total 25
Table (3.8): Thermocycler program for PCR amplification of the CYP2C19*2 and
CYP2C19*3
Step Temperature (ºC) Time Cycles
Initial denaturation 94 3 m 1
Denaturation 94 30 s
37 Annealing 52 30 s
Extension 72 40 s
Final extension 72 5 m 1
4 Hold
3.10.2. Restriction fragment length polymorphism (RFLP) CYP2C19*2
PCR product by Sma1 restriction enzyme
The resulting 321-bp product of CYP2C19*2 PCR was digested in a 20μl reaction
mixture as depicted in Table 3.9. The reaction mixture was incubated at 25 °C for 1 h
followed by an inactivation step at 75 °C for 15 minutes. The digestion products
were analyzed by electrophoresis in a 2.5 % agarose gel and compared to 100 pb
DNA ladder.
29
Table (3.9): The enzymatic digestion components of amplified CYP2C19*2
Solutions Volume Final conc.
Restriction enzyme, Sma1 (20 u/ micro) 0.3 µl 0.3 u/ micro
10xbuffer 2 µl 1X
PCR product 13 µl 13
H2O 5 µl
Total 20 µl
Sma1 cuts the 321-bp PCR products containing the wild type allele into 212-bp and
109-bp fragments and does not cut PCR products containing the mutant allele. The
CYP2C19*2 homozygotes should yield one band (321 bp) while the CYP2C19*2
heterozygotes should produce three bands (321 bp, 212 bp, and 109 bp)
3.10.3. Polymerase chain reaction (PCR) for CYP2C19*3
PCR for the CYP2C19*3 polymorphism, was carried out in a 25 µl reactions, as
described in (Table 3-10). The reaction components were combined in 0.2 ml PCR
tubes. The tubes were placed in a thermocycler and PCR amplification was
performed according to the same program of CYP2C19*2 provided in (Table 3-8).
Table (3.10): PCR components for amplification of CYP2C19*3
Reagent Volume (μl) Final concentration
GoTaq® Green Master Mix (2X) 12.5 1X
CYP2C19*3-F 1 0.2 pmol/µl
CYP2C19*3-R 1 0.2 pmol/µl
DNA 3 About 100 ng in total
Nuclease free water 7.5
Total 25
30
3.10.4. Restriction fragment length polymorphism (RFLP) of CYP2C19*3
PCR product by BamH1 restriction enzyme
The resulting 272-bp product was digested in 30μl volumes with BamH1 as indicated
in Table 3-11. The restriction enzyme was allowed to work at 37 °C for 3 hrs, and
then inactivated at 70 °C for 15 minutes. The digestion products were analyzed by
electrophoresis on a 2.5% agarose gel and identified by comparison to 100-bp DNA
ladder. BamH1 does not cut the PCR products containing the mutant allele but it cuts
the PCR products containing the wild type allele into 175bp- and 96-bp fragments.
The CYP2C19*3 homozygote should yield one band (271 bp) while the CYP2C19*3
heterozygotes should produce three bands (271 bp, 175 bp, and 96 bp).
Table (3.11): The enzymatic digestion components of amplified CYP2C19*3
3.11. Interpretation of CYP2C19 alleles
Each of the two variant allele is caused by a substitution of one nucleotide in turn a
substitution of the amino acid (Table 3-12), leading to different effect in the
enzymatic activity (Table 3-13).
Table (3.12): Interpretation of CYP2C19 alleles
Allele Nucleotide
substitution Amino acid substitution
CY2C19*1 wild type
CY2C19*2 c.681G>A (rs4244285) Cryptic splice acceptor activation
CY2C19*3 c.636G>A (rs4986893) Stop codon gained (nonsense
codon)
(Danielson, 2002)
Solutions Volume Final conc.
Restriction enzyme, BamH1 (20 u/ mico 0.3 µl 0.3 u / micro
10xbuffer 2 µl 1 x
PCR product 13 µl
H2O 15 µl
Total 30 µl
31
Table (3.13): Predicted metabolism phenotypes for CYP2C19 based on example
genotypes
Genotype Predicted metabolism phenotype
CYP2C9-1*/1* Extensive metabolizer (EM)
CYP2C9-1*/2* Intermediate metabolizer (IM)
CYP2C9-1*/3*
CYP2C9-2*/2*
Poor metabolizer (PM) CYP2C9-2*/3*
CYP2C9-3*/3*
(Danielson, 2002)
3.12. Quality control
Negative control samples were applied in each run of PCR along with patients
samples without addition of any DNA material but using a nuclease free water
instead. This blank control served to confirm the quality of testing procedure for
PCR, and the purity of the reagents and lack of contamination. A random sample of
genomic DNA was used as positive controls for the restriction enzyme SmaI and
BamHI, to confirm the enzyme activity and its ability to cut DNA.
3.13. Statistical analysis
The IBM SPSS software version 22, and Microsoft Excel software were used to
perform all the analyses and charts. Descriptive statistics were reported as absolute
frequencies and percentages for qualitative data. In the case of expected frequencies,
more than 5 Chi-square were used and if less than 5 the Fisher’s exact test in the case
of expected frequencies less than 5. All the statistical tests were two sided; a p value
of <0.05 was considered as statistically significant. Multivariate analysis, correlation
coefficient, odds ratios, p value (two-sided tests) and 95% CI were used to describe
the strength of association.
Chapter Four
Results
32
Chapter 4
Results
4.1. Characteristics of the study population:
4.1.1. Personal characteristics
The study enrolled 110 patients, 73 of them were males (66.40%) and 37 were
females (33.60%; Table 4-1). The mean age of the patients was 60.2 ± 11.2, ranging
from 34 to 88 years. The mean age of males was 58.2 ± 11.3 years and that of
females was 64 ± 10 years. According to the residence area, the Northern area has
the lowest frequency 7 of the cases representing 6.5%, followed by the Middle area
with 19 cases (17.3%), Gaza strip with 33 patients (30%), and the Southern area
(Rafah and Khanyouns) with 51 patients (46.4%). Distribution of participants by
education level shows that 32/110 (29.1%) were illiterate, 47/110 (42.7%) were with
school level education, 30/110 (27.3%) were with university level and only one case
(0.9%) with postgraduate education. The average BMI for both genders was 29.3 ±
4.3, of them females had higher BMI than males with mean 32.5 ± 4.1 and 27.71 ±
3.5 respectively.
Table (4.1): Personal characteristics of the study population
Characteristics Frequency % or ±SD
Gender Male 73 66.4%
Female 37 33.6%
Age (Year) 110 60.2 ± 11.2
Education level
Illiterate 32 29.1%
School level 47 42.7%
University
level 30 27.3%
Postgraduate 1 0.9%
Resident area
North Gaza 7 6.5%
Gaza city 33 30%
Mid-Zone 19 17.3%
South Gaza 51 46.4%
Body mass index
(kg/m2)
Male 73 27.7 ± 3.5
Female 37 32.5 ± 4.1
33
4.1.2 Clinical characteristics
4.1.2.1 Cardiac and other chronic diseases
In addition to the cardiac disease, 65.5 % of the enrolled patients had diabetes
mellitus and 69.1% had hypertension (Table 4-2). More than half (60%) had no
family history of heart disease.
Table (4.2): Comorbid chronic disease in the study population
Frequency (%)
Yes No
Diabetes mellitus 72 (65.5%) 38 (34.5%)
Hypertension 76 (69.1%) 34 (30.9%)
4.1.2.2. Complete blood count of the study population
Complete blood count result for the study population were in the normal range for
both genders (Table 3.4).
Table (4.3): Complete blood count of the study population
Mean ± SD
WBC (*109/L) 8.8 ± 2.5
RBC (106/µL) 4.34 ± 0.6
Hb (mg/dl) 12.6 ± 3.9
HCT( %) 38.4 ± 20.5
PLT (103/ µL) 236.8 ± 74.7
WBC= White Blood Cell, RBC= Red Blood Cell, Hb= Hemoglobin, HCT=
Hematocrit, PLT= platelet.
4.1.2.3 Angiographic outcomes
As presented in (Table 4-4), the most frequent lesion found with angiography was in
the left anterior descending artery (LDA) followed by the right coronary artery
(RCA), the left circumflex artery (LCX), the obtuse marginal (OM) and finally the
ramus circumflexes (RCX). Bare metal stents (BMS) were instilled in 96.4% of the
patient, while drug eluting stent (DES) were instilled only in 3.6% patients (Table 4-
4).
34
Table (4.4): lesion location and type of instilled stent
Type of stent Frequency Percent
Lesion location
LDA 49 44.5%
LCX 19 17.3%
OM 6 5.5%
RCA 31 28.2%
RCX 5 4.5%
Stent integrity BMS 106 96.4%
DES 4 3.6%
BMS= Bare Metal Stent, DES= Drug Eluting Stent, LAD= Left Anterior
Descending Artery, LCX= Left Circumflex Artery, RCA= Right coronary artery,
RCX= Ramus Circumflexes, OM= Obtuse Marginal.
4.2. PCR and RFLP results for CYP2C19
4.2.1. CYP2C19*2 PCR product gel electrophoresis
After running on 2% agarose gel the PCR product of CYP2C19*2 was identified as
321 bp DNA fragment using 100 bp DNA ladder along with negative control as
shown in Figure 4.1.
Figure (4.1): PCR products of CYP2C19*2&*3
L=100 bp ladder, lanes 1-7 amplified CYP2C19*2 fragment (321 bp)= Lane 9 - 15
amplified CYP2C19*3 fragment (272 bp), lanes 8 and 16= blank negative control.
CYP2C19*2 CYP2C19*3
35
4.2.2. CYP2C19*2 restriction analysis gel electrophoresis
The PCR product for CYP2C19*2 was introduced to restriction analysis with the
restriction enzyme Sma1. It cut the wild type allele containing 321bp (CYP2C19*1)
into 212-bp and 109-bp fragments and did not cut PCR products containing the
mutant allele (CYP2C19*2). When running on agarose gel CYP2C9*1 shows two
bands while CYP2C19*2 shows one band and the heterozygous shows three band
(Figure 4-2).
Figure (4.2): Restriction products for CYP2C19*2 by SmaI.
L: 100 bp ladder; lanes 1,3 & 4: Normal Homozygote fragment 212 and 109 bp;
Lane 3: Heterozygote fragment 321, 212 and 109bp; lane 5: blank negative control.
4.2.3. CYP2C19*3 PCR product gel electrophoresis
After running on 2% agarose gel the PCR product of CYP2C19*3 was identified as
272 bp DNA fragment using 100 bp DNA ladder along with negative control as
shown in Figure 4-1.
36
4.2.4. CYP2C19*3 restriction analysis gel electrophoresis
The PCR products for CYP2C19*3 were introduced to restriction analysis with the
restriction enzyme BamH1. It cuts the 272bp wild type allele (CYP2C19*1) but does
not cut PCR products containing the mutant allele (CYP2C19*3). When running on
agarose gel restriction digestion of a homozygote CYP2C19*1 gives two bands (175
bp and 96 bp), a homozygote CYP2C19*3 gives one band (272bp) and the
heterozygous gives three band (272bp, 175bp and 96bp) as shown in Figure 4.3.
Figure (4.3): Restriction products for CYP2C19*3 by BamH1.
L: 100 bp ladder; lane 1,2,3 and 4: Normal Homozygote fragment 175-bp and 96-
bp, lane 5: a blank negative control.
4.3. The CYP2C19 Genotyping results
4.3.1. Genotyping frequency of CYP2C19*2 allele
Table 4-5 illustrates the genotype frequencies of CYP2C19*2. The frequency of the
wild type homozygtes (GG) was 70.0 %, the heterozygotes (GA) was 29.1% while
the frequency of the homozygotes for the polymorphic allele (AA) was 0.9%.
CYP2C19*3
37
Table (4.5): Genotyping Frequency of CYP2C19*2 allele
Genotype of CYP2C19 Frequency %
(*1/*1) GG 77 70.0
(*1/*2) GA 32 29.1
(*2/*2) AA 1 0.9
4.3.2. Genotyping frequency of CYP2C19*3 allele
Table 4-6 illustrate the genotype frequencies and percent of Cyp2C19*3. The
frequency of the wild type GG was 95.5 %, the heterozygote GA was 4.5% and no
homozygotes for the polymorphic allele AA were found.
Table (4.6): Frequency of genotypes of CYP2C19*3 allele
Genotype Frequancy %
(*1/*1) GG 105 95.5
(*1/*3) GA 5 4.5
(*3/*3) AA 0 0.0
4.3.3. Overall genotype distribution of CYP2C19
The results of the study indicate that more half of study population (67.3%) were
homozygotes for the wild type allele (*1/*1); 27.3 % were heterozygotes to the
CYP2C19*2 allele (i.e. 1*/2*); 2.7% were heterozygotes to the CYP2C19*3 allele
(1*/3*); 1.8% were compound heterozygotes (2*/3*) and 0.9% homozygotes for
2*/2* mutant allele. On the other hand, the *3/*3 genotype was not detected in the
present study population. By assuming random mating of population in Gaza strip
and applying the Hardy-Weinberg equation for three alleles, the allelic frequency of
the wild type allele *1 was 82.3%, while the frequency of the polymorphic allele *2
was 15.5% and that of the polymorphic allele *3 was 2.3%. The three alleles are in
equilibrium and their frequency is not significantly different from the expected
Hardy-Weinberg frequency (p-value = 0.325; Table 4.7). The calculated chi-square
of our data at 0.05 significant level is 4.69, and the critical value from the chi-square
test table is 7.815. So our calculated chi test is less than critical value from the table,
indicating that our population is in Hardy– Weinberg equilibrium.
38
Table (4.7): Genotype distribution of CYP2C19
Genotype Observed Frequency ¥Expected frequency % by
Hardy–Weinberg law P_value
(*1/*1) 74 74.46
0.325
(*1/*2) 30 27.97
(*1/*3) 3 4.11
(*2/*2) 1 2.63
(*2/*3) 2 0.77
(3*/*3) 0 0.06 ¥ the expected frequency was calculated based on Hardy–Weinberg law
4.3.4. Distribution of CYP2C19 genotypes frequency by gender
The number of male cases in this study was 73 cases, of them, 63% (46/73) were
carrying the wild type allele (*1/*1), 30.1% (22/73) were (*1/*2), 4.1% (3/73) were
(*1/*3) and 2.7% (2/73) were compound heterozygotes (*2/*3) (Table 4.9). The
calculated allele frequency of CYP2C19*1, *2 and *3 alleles in males was found
80.1%, 16.1% and 3.1% respectively. The females were 37 cases, of them, 76%
(28/37) were carrying the (*1/*1) genotype, 21.6% (8/37) were carrying the (*1/*2)
genotype and only one case homozygotes to the *2 allele (*2/*2). The calculated
allele frequency of CYP2C19*1, and *2 alleles in females was 86.5% and 13.5 %
respectively. No *3 allele was found in the females in this study population. The
statistical analysis of shows no statistically significant difference in distribution of
the CYP2C19 genotypes between males and females (P-value 0.332; Table 4.8).
Table (4.8): Distribution of CYP2C19 genotypes by gender
Gender CYP2C19
Total P-value *1/*1 *1/*2 *2/*2 *1/*3 *2/*3
Male 46 22 0 3 2 73
0.332 Female 28 8 1 0 0 37
Total 74 30 1 3 2 110
39
4.3.5 The CYP2C19 genotype and predicted phenotype
According to the genotype status, the metabolizing status of CYP2C19
polymorphism may be classified into three phenotypes, extensive metabolizer (EM)
carrying normal function (wild-type) alleles (CYP2C19*1/*1), intermediate
metabolizer (IM) carrying one loss-of-function allele (*1/*2 or *1/*3) and poor
metabolizer (PM) carrying two loss-of-function alleles (*2/*2, *2/*3 or *3/*3). In
the present study the distribution of patients by the CYP2C19 phenotypes was: 67%
EM, 30% IM and 3% PM (Figure 4.4).
Figure (4.4): CYP2C19 metabolizing status in the study population
4.4 Stent restenosis
Overall, 9 out of the 110 patients enrolled in this study (8.2%) experienced at least
one event of stent restenosis (Figure 4.5). Six of them were carrying the *1/*2
genotype, one case had the *1/*3 genotype, one case the *1/*1 genotype and one
case had the *2/*2 genotype (Figure 4.6).
Figure (4.5): distribution of patient according to restenosis
EM 67.3%
IM 30%
PM 2.7%
9
101
0
20
40
60
80
100
120
Yes No
40
Figure (4.6): Genotyping distribution of restenosis cases
4.4.1. Impact of CYP2C19 polymorphism on stent restenosis
4.4.1.1. Impact of CYP2C19*2 mutation on stent restenosis
The distribution of cases with stent restenosis event by CYP2C19*2 genotype is
presented in Table 4.9. Most of the restenosis cases (7/9) were either hetero- or
homozygous for the polymorphic allele *2. This distribution was found statistically
significant ( (1) = 10.65, P-value = 0.001) [5% (CI), .019-0.5, p=0.001].
Table (4.9): Distribution of restenosis cases by the CYP2C19*2 allele genotype
Genotype Restenosis P_Value
Yes No
10.65 0.001 (*1/*1) 2 75
# (*1/*2) or (*2/*2) 7 26
Total 9 101 #(*1/*2) & (*2/*2) were combined together in one category for the sake of statistical
analysis
4.4.1.2 Impact of CYP2C19*3 mutation on stent restenosis
In contest of the CYP2C19*3 allele genotypes, there are only five cases carrying the
(1*/3*) genotype representing 4.5% of all enrolled cases in the present study. Of
these heterozygotes, only one case suffered from restenosis, representing 11.1% (1/9)
of the restenosis cases (Table 4.10). This distribution was found statistically not
significant (p-value = 0.324).
(*1/*1) (*1/*2) (*1/*3) (*2/*2)
Frequency 1 6 1 1
0
1
2
3
4
5
6
7
41
Table (4.10): Distribution of restenosis cases according to CYP2C19*3 genotype
Genotype Restenosis P_Value
Yes No
0.324 *1/*1 8 97
*1/*3 1 4
Total 9 101
4.4.2. Relationship between restenosis and hypertension
Out of the nine patients who developed restenosis, 7 were hypertensive (77.8%) and
the rest were non-hypertensive. This distribution was statistically not significant
(P=0.556; Table 4.11).
Table (4.11): Distribution of stent restenosis patients by hypertension
Restenosis P_Value
Yes (%) No (%) Total
0.556 Hypertension Yes 7 (77.8) 69 (68.3) 76
No 2 (22.2) 32 (31.7) 34
Total 9 101 110
4.4.3. Relation between restenosis and Diabetes mellitus
Seven cases of the restenosis patients were diabetic (77.8%) compared to 64.4% of
the patients with no restenosis. This difference however, was not statistically
significant (p-value = 0.417; Table 4.12).
Table (4.12): Distribution of stent restenosis patients by diabetes mellitus
Restenosis P-Value
Yes (%) No(%) Total
0.417 DM Yes 7 (77.8) 65 (64.4) 72
No 2 (22.2) 36 (35.6) 38
Total 9 101 110
4.4.4. Relation between restenosis and age
The mean age for restenosis cases was 54.6 ± 9, and for non-stenosis cases was
60.7±11.3 years, The statistical analysis shows no statistical significance in this
distribution (p-value = 0.115 Table 4.13).
42
Table (4.13): Distribution of stent restenosis patients by age
Restenosis P-Value
Yes No
0.115 Mean Age (year) 54.6 ± 9 60.7 ± 11.3
Total 9 101
4.4.5. Relation between restenosis and gender
Out of 110 patients, 73 were men. Similarly, of the 9 patients who developed
restenosis, 8 were men (11%) and one was a woman (2.7%). However, this higher
rate of restenosis in men than in women, after the percutaneous treatment was
statistically not significant (P-value = 0.136; Table 4.14).
Table (4.14): Distribution of stent restenosis patients by gender
Restenosis P-Value
Yes (%) No(%) Total
0.136 Gender Male 8 (11) 65 (89) 73
Female 1 (2.7) 36 (97.3) 37
Total 9 101 110
4.4.6 Relation between restenosis and body mass index (BMI)
The BMI mean for restenosis cases was 27 ± 2.6 kg/m2, and for non-stenosis cases
was 29.5 ± 4.4 kg/m2. The statistical analysis shows no significance relation in this
distribution (p-value = 0.103, Table 4.15).
Table (4.15): Distribution of stent restenosis patients by BMI
Restenosis P-Value
Yes (kg/m2) No (kg/m
2)
0.103 BMI 27 ± 2.6 29.5 ± 4.4
Total 9 101
43
4.4.7 Relation between restenosis and family history of CAD
As indicated in Table 4.16 the 9 patients who developed restenosis, 1 (11.1%) had a
positive family history of CAD. There was no significant relationship between
family history and risk of restenosis (P=0.062).
Table (4.16): Distribution of stent restenosis patients by Family history of heart
disease
Restenosis P-Value
Yes (%) No(%) Total
0.062 Family history
of heart disease
No 8 (88.9) 58 (57.4) 65
Yes 1 (11.1) 43 (42.6) 44
Total 9 101 110
4.4.8 Relation between restenosis and Segment of coronary legion
Out of 110 patients, in-stent restenosis occurs in two arteries, 4/9 cases in the LAD
artery (44.5%) and 5/9 cases RCA artery (55.5%). There was no statistically
significant relationship between the type of lesion and restenosis (P=0.343; Table 4-
17).
Table (4.17): Relation of stent loci and in-stent restenosis
Restenosis P-Value
Yes (%) No(%) Total
0.343 Stent loci
LAD 4(44.4) 45(41.6) 49
RCA 5(55.6) 31(40.7) 36
LCX 0 19 (18.8) 19
OM 0 6 (5.9) 6
Total 9 101 110
LAD= Left Anterior Descending Artery, LCX= Left Circumflex Artery, RCA= Right
Coronary Artery, RCX= Ramus Circumflexes, OM= Obtuse Marginal.
Chapter Five
Discussion
44
Chapter 5
Discussion
Cardiovascular diseases are globally the killer number one, representing 31% of all
global deaths in 2012. Of these deaths an estimated 7.4 million were due to coronary
heart disease (WHO, 2015 and Mathers et al., 2008). The current standard
management for CAD is a dual antiplatelet therapy (aspirin and clopidogrel), in
addition to coronary reperfusion or revascularization (Husted , 2015; Ferreiro and
Angiolillo, 2012). Knowledge of the individual’s genotype can be used to optimize
clopidogrel antiplatelet therapy (Hagymási et al., 2011). The pharmacodynamic
response to clopidogrel varies widely from subject to subject, and about 25% of
patients treated with standard clopidogrel doses display low inhibition of ADP-
induced platelet aggregation (Matetzky et al., 2004). This poor response to
clopidogrel is associated with an increased risk of recurrent ischemic events (Hulot et
al., 2006).
Genetic polymorphisms in CYP2C19, an enzyme required for clopidogrel
bioactivation have been shown to be associated with clopidogrel antiplatelet
effectiveness, and represents a risk factor for recurrent ischemic cardiac events
(Nakata et al., 2013). The polymorphism associated with cloidogrel therapy exhibits
marked racial heterogeneity, with the PM phenotype representing 13-23% of oriental
populations, but only 2-5% of Caucasian populations. Two defective CYP2C19
alleles (CYP2C19*2 and CYP2C19*3) have been described, which account for more
than 99% of Oriental PM alleles and approximately 87% of Caucasian PM
alleles (OMIM, 2016).
To the best of our knowledge, this is the first study in Palestine reporting the impact
of CYP2C19 loss-of-function alleles, CYP2C19*2 and CYP2C19*3, in association
with stent restenosis in CVD patients managed with PCI and clopidogrel as
antiplatelet therapy.
The importance of this study relates to the fact that an understanding of the
distribution of SNPs is crucial for the future application of pharmacogenomic to
45
different population groups. Such applications have recently been performed in
warfarin therapy (Ross et al., 2010).
The small number of patients who experienced stent restenosis (n=9) may have
resulted from the fact that they were given 300 mg of clopidegrel prior to the PCI
and maintained at 75 mg/day for the six later months. Administration of 300 mg
loading dose immediately before PCI is sufficient to prevent major adverse cardiac
events (MARC) (Wolfram et al., 2006). One-third of patients however, may have a
suboptimal antiplatelet response early after intervention using this treatment regimen
(Gurbel et al., 2003).
In this study, the frequency of CYP2C19*1, *2 and *3 alleles was 82.3%, 15.5% and
2.3% respectively. The allelic frequency of CYP2C19*2 is lower than those reported
in Oceanian 61%, South/Central Asian 34%, East Asians 29%, Indian 34% and
Chinese 31% (Xie et al., 2013; Shalia et al., 2013 and Scott et al., 2011). On the
other hand, the frequency of CYP2C19*2 allele was found consistent with Middle
Eastern populations including the Egyptian (11.0%), Saudi (11.2), Lebanese (13%)
Jordanian (16%) , Israeli Jewish (15%), and Tunisian (11.5%); and with African
American (12%), Turkish (13.5%), Iranian (14%), and other Caucasians and
European populations (15%) (Nassar et al., 2014; Abid et al., 2013; Saeed and Mayet
2013; Strom et al., 2012; Zalloum et al., 2012; Jureidini et al., 2011; Scott et al.,
2011; Zand et al., 2005; Hamdy et al., 2002 and Sviri et al., 1999). CYP2C19*2
frequency in our population was slightly different from previous data in Gaza strip,
which showed lower frequency of CYP2C19*2 allele 9.6% and 5.7% among
pediatric hematological malignancy and healthy controls respectively (Sameer et al.,
2009). It was also different from 9.5% that in West bank and Jerusalem Palestinian
population (Nassar et al., 2014). The Palestinian population of Gaza strip may be
considered an inbreeding population because of the political disconnection of the
strip from other Palestinian areas, and the cultural tendency of the community to
mate in a consanguineous way. These factors may contribute to frequency difference
between our study population and west bank Palestinian population.
The CYP2C19*3 allele frequency in our study population is higher than those
recorded in Egyptian (0.2%) and Israeli (1%) (Sviri et al., 1999 and Hamdy et al.,
46
2002), and lower than those reported in Asians (8%), Oceanian 15% (Scott et al.,
2011). On the other hand, this allele was not detected in Belgian, Beninese,
Jordanian, Saudi and Iranian (Al-Jenoobi et al., 2013 and Shalia et al., 2013; Scott et
al., 2011; Zand et al., 2005; Allabi et al., 2003). Our result is consentient with
previous data from Gaza strip (3%) reported by Sameer et al., (2009).
The distribution of CYP2C19 variants in the Palestinian population is consistent with
the relatively high frequency of CYP2C19*2 allele all around the world. Buzoianu et
al., 2010 suggested that this mutation is old and has occurred before the separation of
Caucasians, Oriental and Black populations. By contrast, the CYP2C19*3 allele,
which was very low in our population, is specific to Asian and Oceanian ethnical
groups. The low frequency, or sometimes absence of this allele in different
Caucasians populations confirms the Asian specificity of this mutation and suggests
that this allele occurred quite recently, after the differentiation of Caucasian and
Oriental groups (Buzoianu et al., 2010).
According to the present study the PM phenotype frequencies in Palestinian patients
were 2.7 % which is in consistence with the range reported in Caucasians (2 to 5%)
and lower than in Oriental populations 13-23% (Tassaneeyakul et al., 2002 and
Goldstein et al., 1997).
Despite the small number of patients with stent restenosis, our findings show that
there is a significant relation between stent restenosis and carrying the variant allele
CY2C19*2 ( (1) = 10.65, P-value = 0.001) [5% (CI), .019-0.5, p=0.001]. Similar
results were previously presented by Sibbing et al (2009) who also stated that
CYP2C19*2 carrier status is significantly associated with an increased risk of ST
following coronary stent placement. In other words, we may assume that
the CYP2C19*2 genotype accounted for approximately 6.4% (7/110) of the variation
in clopidogrel response among the present study population.
Our data provides further support to previous reports linking the
CYP2C19*2 genotype and ADP-stimulated platelet aggregation in response to
clopidogrel (Hulot et al., 2006 and Yamamoto et al., 2011).
47
The CYP2C19*3 allele is the second common type among the reduced-function
genes. Our findings however, indicated that there is no significant relation between
stent restenosis and carrying this variant allele (P= 0.324).
Many other factors may exist and favor ST besides genetic polymorphism of
CYP2C19. They include DM, Hypertension, gender, age and BMI.
Patients with DM have higher cardiovascular poor predictor outcomes in all modes
of cardiovascularization (Mathew et al, 2004; West et al, 2004 and Bach et al., 1994).
Coronary in-stent restenosis after PCI occurs more frequently in diabetic individuals
than in non-diabetics (Scheen and Warzée ., 2004). However, our result show no
statistically significant relation between diabetic patient and stent thrombosis after
PCI (P = 0.417). These results are contradicted by a meta-analysis data from 55
studies including 128,084 patients which suggested there was a remarkable negative
effect of DM on coronary stent implantation (Qin et al., (2013), and supported by
meta-analysis which showed that the apparent effect of diabetes on restenosis rates
published in the literature was overrated and reduced to approximately one-half after
adjusting for the difference in age (Gilbert et al, (2004). Also work of Mohan and
Dhall, (2010), suggested that diabetes is not a strong predictor of restenosis.
Out of the nine patients who developed restenosis in this study, 7 were hypertensive.
This distribution was statistically not significant p-value= 0.556. As stated by others,
lack of negative impact of hypertension may result from the significant improvement
of the autonomic nerve function and ambulatory blood pressure indices of patients
with hypertension and coronary heart disease after PCI (yang et al., 2015). On the
contrary, hypertension was proposed by others to be an important predictor of
restenosis (Bach et al., 1994 and Mohan and Dhall, 2010).
Previous studies examining gender differences in patients undergoing coronary
angioplasty have reported that women had a higher in-hospital mortality and were at
increased risk for an adverse outcome in comparison with men (Kahn et al., 1992;
Bell et al., 1993; Watanabe et al., 2001 and Watanabe et al., 2001). On the other
48
hand, this relationship was claimed by other researchers not to exist, and sex and age
were not found to be predictors of restenosis (Jacobs et al., (2002) and Mohan and
Dhall, (2010), Macdonald et al., (1990). Our results advocate no relation between
gender and age and restenosis (p-value= 0.631 and 0.115 respectively).
The BMI mean for restenosis cases was 27 ± 2.6 kg/m2, and for non-stenosis cases
was 29.5 ± 4.4 kg/m2. The statistical analysis, shows no significance relation in this
distribution P-value = 0.103, which consistence with result obtained by Mohan and
Dhall, 2010). Wang et al., (2015) found that there is relationship between increase
the BMI and repeat revascularization for patients who underwent PCI.
Ou of the nine patients who developed restenosis, 4 developed restenosis in the LAD
and 5 in the RCA artery, and no restenosis occurs in the LCX and OM arteries. There
was no statistically significant relationship between the type of lesion and restenosis
(P=0.26).
Chapter six
Conclusions &
Recommendations
49
Chapter 6
Conclusions and Recommendations
6.1. Conclusions
The present study focused on the polymorphisms of CYP2C19 gene in 110 unrelated
men and women patients managed with PCI and treated with Clopidogrel in Gaza
strip- Palestine. The following conclusions can be drown from the study outcomes
The allelic frequencies of CYP2C19 alleles*1 (82.3%), *2 (15.5%) and *3
(2.3%) and frequency of PM 2.7% are in accordance with other Caucasian
populations, while none of the enrolled subjects was homozygous for
CYP2C19*3.
A significant relationship was found between in-stent restenosis, found in
8.2% of patients and carrying the CYP2C19*2, but not the CYP2C19*3
allele.
Other risk factors for in-stent restenosis including hypertension, diabetes
mellitus, age, sex and body mass index were not found to be significant
relation in our populations.
50
6.2. Recommendations
The data presented in this study will help physicians prescribing clopidogrel or
considering other antiplatelet therapy by providing an approximate percentage of
poor metabolizers. It is therefore recommended to:
Test the polymorphisms CYP2C19*2 and CYP2C19*3 for patients with in-
stent restenosis. This would allow physicians to give a genotype-guided
treatment, which could be more cost-effective and result in a reduction of
clinical adverse outcomes.
Further studies must be performed to evaluate the role of other alleles
CYP2C19 genes as possible determinants recurrent of stenosis.
51
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Annex (1): Approval of Helsinki committee
66
Annex (2): Approval of MOH for sample collection
67
Annex (3): Data collection
Personal
Data……………………………………….. Date ……………………………….
Name
(optional) ………………………. Age ……. Hospital Number ………..
Mobile ……………………….. Gender ……. Residence Area ……….
Weight ……………………… Height …… Education level ………..
Medical Data
Surgical Operation
Yes No Years # Comments
PCI (stand) ……. …. …… …. …………………………
Open Heart Surgery …… …. ……. …. ……………………………
Medication
Yes No dose day Period
Clopidogrel ….. ….. …….. ……… …………………………………
Aspirin ….. ….. …….. ……… . ………………………………
Laboratory data
WBC ………………….
RBC ………………….
Hb ………………….
PLT ………………….