University of Southern California Dissertations and Theses

Analysis of the ALOX5 gene in atherosclerosis

(USC Thesis Other)

Analysis of the ALOX5 gene in atherosclerosis

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content ANALYSIS OF THE ALOX5 GENE
IN ATHEROSCLEROSIS
By
Xuemei Deng
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
May 2005
Copyright 2005 Xuemei Deng
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
UMI Number: 1427970
INFORMATION TO USERS
The quality of this reproduction is dependent upon the quality of the copy
submitted. Broken or indistinct print, colored or poor quality illustrations and
photographs, print bleed-through, substandard margins, and improper
alignment can adversely affect reproduction.
In the unlikely event that the author did not send a complete manuscript
and there are missing pages, these will be noted. Also, if unauthorized
copyright material had to be removed, a note will indicate the deletion.
®
UMI
UMI Microform 1427970
Copyright 2005 by ProQuest Information and Learning Company.
All rights reserved. This microform edition is protected against
unauthorized copying under Title 17, United States Code.
ProQuest Information and Learning Company
300 North Zeeb Road
P.O. Box 1346
Ann Arbor, Ml 48106-1346
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
DEDICATION
I will dedicate this thesis to my mother, who is now in heaven. But I still want
to say thank you mom. You mean more to me than words will ever be able to express
and I just wish I had told you these things.
I will dedicate this thesis to my daughter. I want to express how I love you,
Yuanyuan.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
ACKNOWLEGMENTS
I would like to take this opportunity to thank Dr. Juergen Reichard, my
advisor, for giving me the opportunity to join his lab. He is a wonderful person and
the greatest scientist I have ever met. His keen observation has been and will
continue to inspire me.
I would like to thank my committee members, Dr.Zolten Tokes for guiding
me during my M.S. I thank Dr. James Ou, for his helpful comments and his time for
reading my thesis.
I would like to thank Dr. Clemente Capasso, who was my personal mentor, for
teaching me all the new techniques. His expertise in genetics and molecular biology
improved my research skills and prepared me for future challenges. I thank Troy
Phipps for always being helpful in my lab work, analysis data and during writing my
thesis.
I firmly believe that the work environment makes the greater part of the
learning experience and for this, I would like to thank my colleagues: Dr. Nick
Makridakis, Albert Kim, Chirag Patel, Hannah Nguyen, and Frank Luh in Dr.
Juergen Reichard’s lab.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
TABLE OF CONTENTS
Dedication ii
Acknowledgements iii
List of Tables vi
List of Figures vii
Abstract viii
1. Introduction 1
1.1 Atherosclerosis 1
1.2 Atherosclerosis Epidemiology 2
1.3 Stages of Atherosclerosis 3
1.3.1 Endothelial Cells Dysfunction and Atheogenesis 3
1.3.2 The Evolution of Atherosclerotic Lesions 6
1.4 History of Human Atherosclerosis 6
1.5 Risk Factors 7
1.5.1 Cholesterol and Lipoprotein 8
1.5.2 Age 9
1.5.3 Males 10
1.5.4 Family History 10
1.5.5 Hypertension 11
1.5.6 Smoking 12
1.5.7 Obesity 12
1.5.8 Diet 13
1.5.9 Homocysteine 13
1.5.10 Oxidative Stress 14
1.5.11 New and Emerging Risk Factors 14
2. The Human 5-Lipoxygenase Gene and Atherosclerosis 16
2.1 Supporting Evidence for the Role of 5-Lipoxygenase
in Atherosclerosis 16
2.1.1 Leukocyte Expression of 5-Lipoxygenase 16
2.1.2 5-Lipoxygenase -Derived Leukotrienes Biosynthesis 17
2.1.3 Inflammatory Effects of Leukotrienes 20
2.1.4 Identification of 5-Lipoxygenase Gene as a Major
Gene Contributing to Atherosclerosis Susceptibility in Mice 20
2.2 The Human 5-Lipoxygenase Gene 21
2.2.1 Structure of the Human 5-Lipoxygenase Gene 22
2.2.2 5-Lipoxygense Protein 22
2.3 ALOX5 Gene Polymorphism and Atherosclerosis 25
iv
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
2.3.1 Genetic Polymorphisms in the ALOX5 Gene and
Cancer Risk 25
2.3.2 Single Nucleotide Polymorphisms (SNPs) and
the ALOX5 Gene 26
2.3.3 Gene Expression of the ALOX5 Gene 27
2.3.3.1 Gene Expression 27
2.3.3.2 Promoter Region and Gene Transcription 27
2.3.3.3 Promoter Region of the ALOX5 Gene 30
2.3.4 Hypothesis 31
3. Materials and Methods 33
3.1 Subjects and DNA Isolation 33
3.2 PCR Amplification of ALOX5 3 3
3.2.1 Primer Design 3 3
3.2.2 Polymerase Chain Reaction (PCR) 36
3.3 Gel Electrophoresis and PCR Product Purification 37
3.4 Automated DNA Sequencing 37
4. Results 39
4.1 Polymerase Chain Reaction (PCR) Results 39
4.2 Purified PCR Products 41
4.3 Mutation Identification 42
4.4 Data Analysis 50
4.4.1 Hardy Weinberg Equilibrium 50
4.4.2 Haplotype Analysis of ALOX5 Gene 52
5. Conclusion and Discussion 57
5.1 Conclusion 57
5.2 Promoter Analysis 58
5.3 Substitution in the 5 ’Untranslated Region (5 ’UTR) 60
5.4 Silent Substitutions 61
5.5 Future Work 62
Reference 64
v
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
LIST OF TABLES
3.1 PCR reagents, volumes and final concentration 36
3.2 Internal primers within the promoter region used
during automated sequencing 38
4.1 Hardy Weinberg equilibrium 54
4.2 Position and frequency of polymorphisms used in
hyplotype analysis 55
4.3 Hyplotype frequencies of ALOX5 gene 56
5.1 TFSEARCH 2.0 search result 60
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
LIST OF FIGURES
1.1 Atherosclerosis in the coronary artery 2
1.2 Five leading causes of death for all males and females 3
1.3 Stages of atherosclerosis 5
1.4 History of human atherosclerosis 7
1.5 Lipoprotein metabolism 9
2.1 Main putative roles of leukotrines in atherosclerosis 18
2.2 The leukotrienes synthetic pathway 19
2.3 Structure of the human 5-lipoxygenase gene 22
2.4 Model of 5-lipoxygenase 24
2.5 Conserved locations in complex eukaryotes for regulatory
promoter elements bound by ubiquitous transcription factors 28
2.6 Promoter region of the ALOX5 gene 31
3.1 PCR primer annealing sites on promoter region,
exons 1,12 and 13 of the ALOX5 35
4.1 PCR products 40
4.2 Purified PCR products 42
4.3 Sequence variants were found in promoter region of ALOX5 47
4.4 Sequence variants were found in 5 ’UTR of ALOX5 48
4.5 Sequence variants were found in exons 1 and 13 of ALOX5 49
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
ABSTRACT
Atherosclerosis is the leading cause of death in the United States, and is
considered a chronic inflammatory process. 5-Lipoxygenase (encoded by the
ALOX5 gene) is a key enzyme in the anabolism of arachidonic acid into leukotrienes
(inflammatory mediators). I hypothesize that genetic polymorphism in the ALOX5
gene may play a role in atherosclerosis development. A mutational analysis of the
ALOX5 gene was carried out. Genomic DNA samples from eighty-six individuals
were PCR amplified and sequenced to find sequence variants. Ten DNA sequence
variants were noted in the promoter region. These variants are: -1753 G— »A,
-1744T— »A, -1700G— >A , 6-base deletion at position -1361 to -1366, -1145G— »C,
-839G— >A, -754C— ► G , -557T— >C, -310A— »G, -59C— >T. Two substitutions -28G— >C
and -15C— »G were identified in the 5’ untranslated region. Two silent substitutions
21C— >T and 1728A— »G were identified in exons 1,12 and 13.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1.0 INTRODUCTION
1.1 Atherosclerosis
Atherosclerosis is a chronic inflammatory disease of large and medium
sized arteries (Ross, 1999). It is characterized by hypertrophy of the vascular
media, intimal thickening and lipid-containing plaques (Wennmalm, 1994). The
arteries are blood vessels that supply blood, oxygen and nutrients to the body
from the heart. As plaque builds up in an artery, the artery gradually narrows and
can become clogged as shown in Figure 1.1
(http://www.mds.qmw.ac.uk/morbidanatomy/intercal/handoutsetc, 2004). Those
parts of the body affected by atherosclerosis suffer the consequences of an
inadequate blood supply, poor function, tissue damage or death (Fuster, 1996).
Atherosclerosis most commonly affects the heart, brain and peripheral
arteries (Fuster, 1996). Coronary artery disease is an imbalance between the
supply and demand of blood for cardiac muscle, and leads to angina, myocardial
infarction, sudden cardiac death, and chronic heart failure (Fallon, 1996). In the
brain, the syndromes mainly associated with atherosclerosis are stroke, transient
ischemic attack and mental deterioration. This may cause irreversible or
reversible injury. Stroke often destroys a region of the brain due to infarction and
hemorrhage (Fuster, 1996). A transient ischemic attack is a "mini-stroke" caused
by temporary disturbance of blood supply to an area of the brain, resulting in a
sudden, brief decrease in brain function. Mental deterioration is due to gradual
1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 1.1 Atherosclerosis in the coronary artery (Reproduced from
http://www.mds.qmw.ac.uk/morbidanatomy/intercal/handoutsetc)
death of brain tissue over many years (Fuster, 1996). Peripheral arterial disease
develops when the arteries in the arms, legs or pelvis are affected. In the legs and
arms, atherosclerosis may cause cramping pain in the muscles on exertion. In the
kidneys, atherosclerosis can lead to renal failure (Glagov, 1996).
1.2 Atherosclerosis Epidemiology
It was reported that cardiovascular diseases are the leading cause of the
illness and death in the United States (Anderson, 2000). Atherosclerosis accounts
for nearly three-fourths of all deaths from cardiovascular diseases which cause
more death each year than the next 4 leading causes of death combined as shown
in Figure 1.2 (American Heart Association, 2004). Furthermore, the Global
Burden of Disease Study predicts that coronary heart disease, the most important
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
clinical manifestation of atherosclerosis, is and will remain the main reason of
death from 1990 to 2020 in developed counties (Murray, 1997).
United States: 2001
4 9 8 ,8 6 3
Maies
Females
268,693
8 6 ,0 8 0
6 3 3 1 6 38,531
A Total CVD D Chronic Lower Respiratory D iseases
B Cancer E Diabetes Mellitus
C Accidents F Alzheimer's D isease
Figure 1,2 Five leading causes of death for all males and females
(Reproduced from Heart and Stroke Statistical Update from American Heart
Association, 2004).
1.3 Stages of Atherosclerosis
1.3.1 Endothelial Cells Dysfunction and Atherogenesis
The initial steps that subsequently lead to atherosclerosis are only
partially known (Braunwald, 1997). According to the “response-to-injury”
hypothesis by Ross, lesions of atherosclerosis could arise as a result of repetitive
injury to the endothelium of the artery (Ross, 1999). This injury could be due to
any number of factors, which are oxidative forms of LDL-cholesterol,
3
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
hypertension, smoking, low HDL, elevated plasma homocysteine concentration
and genetic alteration (Renke, 2003). Endothelial cells are important in acting as
a selectively permeable barrier (Gimbrone, 1981), and the control of coagulation,
fibrinolysis, vascular tone, growth, and immune response (Stenvinkel, 2001). The
normal endothelium does not in general support binding of leukocytes. In
response to injury, endothelium may become activated and change its normal
function. These changes included increasing the endothelial permeability,
providing more location for lipoprotein to deposit, up-regulating the leukocyte
adhesion molecules, and emigrating the leukocytes into the artery wall. In the
vascular wall, macrophages accumulate oxidized LDL cholesterol via scavenger
receptors pathway and become large foam cells. Endothelium, monocytes,
macrophages, foam cells and platelets release growth factors, cytokines,
chemokines. In response to these factors, smooth muscle cells modify, proliferate
and enter intima from the media layer. Macrophages continue to accumulate lipid
and support endothelial cell dysfunction at site of injury. Eventually foam cells,
T cells and smooth muscle cells form the fatty streak as shown in Figure 1.3
(reviewed in Ross, 1999). Fatty streak lesions are the first grossly visible lesion
in the arterial wall (Stary, 1994).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
NORMAL ART6RV
Adventia
FATTY STREA K
Macrophage
Smooth
Foam Cells
Muscie
Intima
Endothelium
EARLY ATHEROMA
Fiorous Gap
Lipio Ricn
Nectrotic
Core „
Large
Nectrotic
C o r6 \
..A..
VULNERABLE PLAQUE
Thin Fibrous Cap
Smali
Lipid
POol
STABILIZED PLAQUE
Thick Fibrous Cap
THROMBOSIS OF A
RUPTURED PLAQUE
Thrombus
Figure 1.3 Stages of atherosclerosis (Modified from Lusis, 2004).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1.3.2 The Evolution of Atherosclerotic Lesions
As fatty streaks progress to intermediate and advanced lesions, they tend
to form a fibrous cap that walls off the lesion from the vascular lumen. This
represents a type of healing or fibrous response to the injury. The fibrous cap
covers a mixture of leukocytes, lipid and debris, which may form a necrotic core.
The necrotic core represents the results of apoptosis and necrosis, increased
proteolytic activity and lipid accumulation. Some plaques are stable and become
calcified, thus “hardening” the artery. But others with a thin fibrous cap,
especially those rich in lipids and inflammatory cells, may undergo rapture.
These plaques are deemed unstable and are more closely associated with the
onset of an acute ischemic event. Each of the stages of lesion formation is
potentially reversible (reviewed in Ross, 1999).
1.4 History of Human Atherosclerosis
Atherosclerosis can start as intimal lipid deposit (fatty streaks) in
childhood adolescence (McGill, 2000). If the cause of injury is removed, fatty
streaks formation is potentially reversible. If continued injury happened, fatty
streaks in some arteries may progress over years. Finally, they trend to form
fibrous cap (McGill, 2000). In the middle age, fibrous plaques undergo a variety
changes, including calcification, hemorrhage, ulceration, thrombosis, some of
which precipitate clinically manifest disease (McGill, 2000). Typically, clinical
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
disease occurs 30 or more years after the fatty streaks, but it can be greatly
accelerated in persons who are exposed in risk factors (McGill, 2000) (Figure
1.4).
c u i m c a i . m m m n
teste*
mmmmmu, urnmmm.
Q
<
f w»mm Hume
M f m S T ftfM
Figure 1.4 History of human atherosclerosis (Reproduced from McGill,
2000)
1.5 Risk Factors
The development of atherosclerosis is a complex process. Many risk
factors have been found to contribute to atherosclerosis, such as age (National
Center for Health Statistics, 2002), being male (Labarthe DR, 1998), family
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
history (Marenberg, 1994), hypertension (Crouse, 1987; Tell, 1989; Salonen,
1991), smoking (Weintraub, 1985), serum cholesterol level (Johnson, 1993),
serum homocysteine level (Patricia, 1997), obesity (Rimm, 1995), and oxidative
stress (Bennett, 2001).
1.5.1 Cholesterol and Lipoprotein
Epidemiological studies have demonstrated a link between the elevated
low density lipoprotein (LDL) cholesterol levels and the increased incidence of
coronary heart disease (Johnson, 1993). Elevated LDL cholesterol, along with
decreased HDL cholesterol, has been shown to promote the development of
atherosclerosis. Conversely, reducing the LDL cholesterol level and raising the
HDL cholesterol level significantly reduces the risk of this disease (Tailleux,
2002). Plasma concentrations of LDL and HDL are related to lipoprotein
metabolism. Figure 1.5 shows an overview of lipoprotein metabolism. A primary
initiating step in atherosclerosis is LDL bound to proteoglycan in the intima. The
trapped LDL undergoes oxidation modification and does taken up by
macrophages via scavenger receptors, resulting in the formation of foam cells.
The trapped LDL is greater when levels of circulating LDL are elevated
(reviewed in Lusis, 2004). On the other hand, HDL is called the "good
cholesterol" because HDL particles prevent atherosclerosis by extracting
cholesterol from the artery walls and disposing it in the liver where cholesterol is
8
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
processed and removed (Gordon, 1977). Hydroxymethylglutaryl coenzyme A
reductase inhibitors (statins) therapy has been shown to reduce cardiovascular
disease events by their LDL-lowing effects (Raul, 2003).
m
* * / m S
oammn.
* s v
tmmm. t i s s u e s
Figure 1.5 Lipoprotein metabolism (Reproduced from Lusis, 2004)
1.5.2 Age
Atherosclerotic lesion begins as fatty streaks in childhood and
adolescence (McGill, 2000). Cardiovascular diseases are the third cause of death
for children under age 15. In 2001, about 197,000 people were diagnosed with
atherosclerosis at 15 years old or younger (American Heart Association, 2004).
9
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Even fatty steak formation has been demonstrated in human fetal aortas from
hypercholesterolemic mothers (Napoli, 1997). Although the atherosclerosis does
happen at a young age, it is more likely to show clinical symptoms (sudden
cardiac death, myocardial infarction, stroke, or peripheral arterial disease) with
age. In 2002, cardiovascular diseases cause 463,801 deaths beyond 75 years old
in the United States (National Center for Health Statistics, 2002).
1.5.3 Males
Being male is one of the strongest risk factors of atherosclerosis
(Labarthe, 1998). Men who currently develop a cardiovascular disease have a
greater risk than women do (Heart disease and stroke statistics, 2005 Update).
The average age of clinical symptoms of coronary artery disease was about 10
years later in women than in men (Rannel, 1976). In premenopausal women,
coronary disease is rare, but increases during and after menopause. Thus the rates
become nearly equal in men and woman at the older ages (Vanecek, 1976). A
possible cause of the sex differences in coronary disease prevalence may due to
hormone differences (Labarthe, 1998).
1.5.4 Family History
Atherosclerosis occurring in younger people usually clusters in families.
Grech studied 387 patients and suggested that family history is an independent
10
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
risk factor for coronary artery disease (Grech, 1992). Risks for coronary heart
disease death are greater in monozygotic compared with dizygotic twins,
particularly when there is a premature (<65 years) age of onset in the initially
affected twin (Marenberg, 1994). Research has documented that many genetic
variants were associated with probability of atherosclerosis. These variations
include a defect in the LDL receptor gene (Descamps, 2001), polymorphisms in
apolipoproteins (Scanu, 1992), and the genes related with other independent risk
factors for atherosclerosis that have a genetic background, such as hypertension,
diabetes, and hyperhomocysteinemia (Lusis, 1988).
1.5.5 Hypertension
In several studies, atherosclerosis has occurred more in people with
hypertension than in controls (Crouse, 1987; Tell, 1989; Salonen, 1991). Recent
advances in the understanding of vascular disease genesis suggest that
atherosclerosis and hypertension may have a common causal mechanism:
endothelial dysfunction (Ceravolo, 2003). Atherosclerosis and hypertension
development also share similar risk factors, such as the protein angiotension II,
which causes the raising ofblood pressure, the endothelial dysfunction and the
development of atherosclerotic plaques (Zhao, 2004). A large number of studies
have proved that antihypertensive treatment can drop both hypertension and
progression of atherosclerosis (Pepine, 2001; Takais, 2003).
11
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1.5.6 Smoking
Epidemiological evidence suggests that smoking increases the risk of
atherosclerosis, and is the major risk factor for cardiovascular mortality
(Weintraub, 1985). The effects of smoking on cardiovascular system are by
several different mechanisms. Firstly, components of smoke including carbon
monoxide, nicotine, polycyclic aromatic hydrocarbons, and other chemicals
increase oxidative stress and cause arterial endothelial dysfunction (Raij, 2001).
Secondly, smoking increases the risk associated with other risk factors like
cholesterol (Ingolfsson, 1994). Thirdly, smoking, even breathing secondhand
smoke increases platelet activity, accelerates atherosclerotic lesions, and
increases tissue damage following ischemia or myocardial infarction (Glantz,
1995). Smoking also increases heart rate, blood pressure, causing reduced blood
flow to the heart (Ball, 1974). Steenland reported that the risk of death due to
cardiovascular diseases increases by 30% in nonsmokers who live with smokers
(Steenland, 1992).
1.5.7 Obesity
Obesity is defined as an excessively high amount of body fat or adipose
tissue in relation to body mass. It is associated with too many adipose cells,
adipose cells being too large, or both (Stunkard, 1993). Obesity is an
independent risk factor for atherosclerosis (Rimm, 1995). Adipose cells
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
increase the secretion of proinflammatory cytokines, such as IL-6 (Mohamed,
1997) and adiponectin (Maeda, 1996). Chronic inflammation may be one
pathophysiological mechanism explaining the increased risk of atherosclerotic
disease associated with adiposity. Obesity also contributes to risk of
atherosclerosis through its effect on hypertension (Pi-Sunyer, 1993).
1.5.8 Diet
Epidemiological studies have shown a correlation between diet and
atherosclerosis. Excessive intake of triglycerides (Durrington, 1998) and
cholesterol can increase average serum cholesterol levels and atherosclerosis
(Keys, 1980). But vegetarian diets reduce atherosclerosis progression
(Segasothy, 1999). An inverse association between soybean, legumes, nuts,
calorie restriction, folic acid, unstaturated fat and antioxidant effects (such as
vitamin E, vitamin C) and the risk of atherosclerosis has been reported
(Segasothy, 1999).
1.5.9 Homocysteine
Elevated plasma homocysteine has been found to be associated with an
increased risk of atherosclerosis (Patricia, 1997). Homocystinuria is an inherited
disorder. Affected individuals do not break down homocysteine and have
abnormally high serum levels of homocysteine. If untreated, the patients often
13
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
die of strokes and heart attacks before reaching adulthood (Blom, 1998). The
current hypothesis is that high serum homocystein enhances LDL oxidation,
stimulates endothelial smooth muscle to grow, and therefore induces the change
in endothelial function and the growth of the atherosclerotic plaques (Kario,
2001). The most common reason for increased homocysteine concentration is
usually from decreased enzymes activity involved in this metabolic pathway.
Hyperhomocysteinemia can also be acquired if people have nutrient deficiencies
in folate, Vitamin B12 or Vitamin B6 (Pietrzik, 1998).
1.5.10 Oxidative Stress
Oxidative stress occurs when reactive oxygen species (ROS) occurs and
exceeds the body’s ability to neutralize and eliminate them. If not properly
regulated, the excess ROS can damage a cell’s lipids, proteins or DNA,
inhibiting normal function (Bennett, 2001). Increased ROS levels may be
involved in four fundamental mechanisms that contribute to atherogenesis:
oxidation of LDL, endothelial cell dysfunction, vascular smooth muscle cells
growth and leukocyte migration (Berliner, 1996). Recent studies showed an
association between increased oxidative stress and hypertension (Berry, 2001),
smoking (Pittilo, 2000) and high serum cholesterol level (Ehara, 2001).
1.5.11 New and Emerging Risk Factors
14
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Risk factors mentioned above for the disease have been identified
through epidemiological studies. However these risk factors do not appear to
fully explain how atherosclerosis occurs in all people. Present experience
suggests that the relative reduction of atherosclerosis risk in tightly managed
patients in clinical trails ranges from 20-40% (reviewed in Renke, 2003).
Therefore, other less well-studied risk factors may also play important roles
(Hopkins, 1981).
Studies in humans and mice have begun to reveal genetic factors that
contribute to atherosclerosis susceptibility independent of traditional risk factors
mentioned above. The recent studies suggest that human 5-lipoxygenase gene
was a possible candidate gene that may play a role in atherosclerosis. Human 5-
lipoxygenase protein is significantly increased in atherosclerosis lesions
(Spanbroek, 2003) and it is the key enzyme for inflammatory mediators’
synthesis (Werz, 2002). Atherosclerosis is considered a chronic inflammatory
process (Ross, 1999). Thus, human 5-lipoxygenase gene polymorphisms may
influence enzyme expression and function. People with a variant form of a gene
may have a greater risk of atherosclerosis.
15
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
2.0 THE HUMAN 5-LIPOXYGENASE GENE AND
ATHEROSCLEROSIS
2.1 Supporting Evidence for the Role of 5-Lipoxygenase in
Atherosclerosis
2.1.1 Leukocyte Expression of 5-Lipoxygenase
It is currently believed that atherosclerosis in the general population
results from a combination of numerous risk factors. In addition to traditional
risk factors, inflammation is now recognized as a major force driving
atherosclerosis (Ross, 1999). Inflammation participates in all stages of
atherosclerosis. In addition to the involvement of inflammatory cells in
atherosclerosis, many proinflammatory mediators such as cytokines and
chemokines lead to responses that promote lesion formation and plaque ruptures
(Hansson, 2002).
Leukocytes are cells in the blood that are involved in defending the body
against infective organisms and foreign substances. The recruitment of blood
leukocytes to the arterial intima is a key feature of the initiation and progression
of atherosclerotic lesions (Watanabe, 1989). During atherogenesis, leukocytes
adhere, aggregate and emigrate to the injured endothelium from the blood.
Monocytes may differentiate into mature macrophages that engulf oxidized LDL
cholesterol and lead to foam cell formation. Moreover, atherosclerotic lesions
have an abundant inflammatory infiltrate, including macrophages (Libby, 1995),
16
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
T cells (Libby, 1995), and mast cells (Laine, 1999) during progression of
atherosclerosis. Infiltrating leukocytes provide the source of a number of
vasoactive mediators and enzymes that help expand the inflammatory response
(Predimank, 2002). Thus monocytes, macrophages, foam cells and mast cells are
important in the atherosclerosis initiation and progression.
5-Lipoxygenase is specifically expressed in monocytes, macrophages,
foam cells, mast cells and neutrophilic granulocytes and the number of enzyme
expressing cells is significantly increased in advanced atherosclerotic lesions
(Spanbroek, 2003). Recently, the understanding on atherosclerosis is as a chronic
inflammatory disease of the vascular wall (Ross, 1999). 5-Lipoxygenase
pathway has been linked to atherosclerosis because it plays important roles in
inflammation as shown in Figure 2.1 (Caterina, 2004).
2.1.2 5-Lipoxygenase-Derived Leukotrienes Biosynthesis
5-Lipoxygenase is the rate-limiting enzyme in the oxidative biosynthesis
of leukotrienes (Werz, 2002). The synthetic pathway for leukotrienes is initiated
by the release of arachidonic acid from the cell membrane by phospholipase A2,
and then the enzyme 5-lipoxygenase converts arachidonic acid into the unstable
LTA4, a central intermediate in leukotrienes synthesis. LTA4 may be hydrolyzed
into LTB4 by the enzyme LTA4, hydrolase or conjugated with glutathione to
form the cysteinyl leukotriene LTC4. The cysteinyl leukotrienes include LTC4
17
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
and its metabolites, LTD4 and LTE4 (reviewed in Werz, 2002). The leukotrienes
synthetic pathway is shown in Figure 2.2.
V.’Jv f ”‘ !
ft)#'?*"''
2 s s , s( ? 'i< I E , jf ifiii
r'
* \
t iM Jtd
p f tin iiliiilv
‘fsfcw
• f liif W S te
f
A
1
.-.dL
N S W
(0m
7
,j s^itefK fesils
7 ( 5 1 ! * '
, 1 * 1 8 ' ■ ■ ■
4 9 0
riMnBWMI
Figure 2.1 Main putative roles of leukotrienes in atherosclerosis
(Reproduced from Caterina, 2004)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
W \ = y \ / * \ /
AracJfidoaic acid
5-Lip eiygaou
+ 0.
COOH
COOH
5-HETE
t C C 0 H
OH
LFAfHydrolase /
i , ra o n tw te y
OH
,COOH
,COOH
R = — C y s - G lv LTQ
I
Glu
R - — C ys— Ch' LTD,
%
R = - C y s LTEj
Figure 2.2 The leukotrienes synthetic pathway (Reproduced from Werz,
2002)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
2.1.3 Inflammatory Effects of Leukotrienes
Leukotrienes are a family of biologically active lipids, synthesized and
released from leukocytes, which have a variety of proinflammatory effects
(Goetzl, 1995).
The studies suggest that LTB4 is a potent chemoattractant for monocytes.
It can promote atherosclerosis by recruiting monocytes to the endothelium of
vessel wall (Bray, 1981). LTB4 receptor null mutant mice had significant defects
in macrophage recruitment (Haribabu, 2000). Using LTB4 receptor antagonism
reduced foam cells formation in mice (Aiello, 2002). In additional, LTD4 causes
autocrine activation of macrophages. Activated macrophages release MIP-1,
which may further promote T cell recruitment during atherogenesis (Zhao,
2004). LTC4 and LTD4 can promote the adhesion and extravasation of
monocytes, and were found to up-regulate surface expression of P-selectin, one
of the cell adhesion molecules active during leukocytes accumulation in
atherosclerosis (Datta, 1995).
Thus given the inflammatory properties of leukotrienes, we reasoned that
genetic variation in the human 5-lipoxygenase gene might promote
atherosclerosis.
2.1.4 Identification of 5-Lipoxygenase Gene as a Major Gene Contributing
to Atherosclerosis Susceptibility in Mice
20
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
An identified locus for atherosclerosis is noted on mouse chromosome 6
(Mehrabian, 2001). B6 mice are an atherosclerosis susceptible inbred strain.
Feeding B6 mice an atherogenic diet develops atherosclerotic lesions in large
arteries. In contrast to the B6 strain (Paigen, 1987). CAST mice are highly
resistant to diet-induced atherosclerosis (Mehrabian, 2001). Mehrabian et al.
first constructed a cross strain called CON6 between CAST mice and B6 mice
(Mehrabian, 2001). By examining the congenic region for potential positional
candidate genes, they found that the human 5-lipoxygenase homologous gene in
mice is located near the middle of the congenic region in chromosome 6. The
level of 5-lipoxygenase mRNA and protein were reduced in an atherosclerosis-
resistant mouse strain CON6, as compared with a susceptible strain B6. In
addition, mice with 5-lipoxygenase knockout allele onto an LDL receptor-null
background were also bred. They showed a dramatic decrease (>26 fold) in
aortic lesion development (Mehrabian, 2002). These experiments indicate that 5-
lipoxygease contributes importantly to the atherogenic process. This also
provides strong presumptive evidence that reduced 5-lipoxygease expression is
partly responsible for the resistance to atherosclerosis.
2.2 The Human 5-Lipoxygenase Gene
The human 5-lipoxygenase (EC 1.13.11.34), encoded by the ALOX5
gene, is a rate-limiting enzyme that catalyzes the two-step conversion of
21
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
arachidonic acid to leukotriene A4 (Werz, 2002) as shown in Figure 2.2. Thus,
the enzyme 5-lipoxygease plays an important role in regulating the amount of
leukotrienes that are known to be involved in atherosclerotic inflammation
(Radmark, 2003). This significant role of 5-lipoxygenase makes it necessary to
detect in detail during the study of atherosclerosis.
2.2.1 Structure of the Human 5-Lipoxygenase Gene
The ALOX5 gene is located on the long arm of chromosome 10
(10qll.2). As shown in Figure 2.3, the ALOX5 gene spans over 82 kb of
genomic DNA and contains 14 exons and 13 introns (Funk, 1989).
u m a 1 1 7 ^ 6 g 1 2 1 1 * * 4 7 l n l * n 7 1 9 9 1 9 0 2 8 7 1 2 2 1
I jSpll __■ / ’
I p ill llllll i B l i § § l l l | i t t l 3 ‘U "[r j
— 3 i -------5 " f> — 7 — 8 ~ ‘i - 1,1 — I i -12-1.1 "11 ✓ ------- I —J
- ; " ■ ^' i
8 2 1 2 3 1 0 7 1 7 3 1 4 7 2 0 4 8 7 1 7 9 1 2 2 1 0 1 1 7 1 6 0 6
Figure 2.3 Structure of the human 5-Iipoxygenase gene
(14 exons in boxes and introns in straight lines between exons)
2.2.2 5-Lipoxygenase Protein
5-Lipoxygenase is a dual function enzyme in the biosynthesis of
leukotrienes. It catalyses the incorporation of molecular oxygen into arachidonic
22
x rts4
1 --- r .
1 9 2 1 9 9
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
acid (oxygenase activity), and subsequently forms of the unstable epoxide LTA4
(LTA4 synthase activity) (Figure 2.2) (Samuelsson, 1983).
Human 5-lipoxygenase protein is composed of 673 amino acids
(Matsumoto, 1988). It has been purified as a monomeric soluble protein with a
calculated molecular weight of 77839 (Radmark, 2000). The crystal structure of
5-lipoxygease has not been solved, but was modeled as shown in Figure 2.4
based on the solved rabbit reticulocyte of 15-lipoxygenase.
5-Lipoxygease is with two domains, the larger one is a catalytic C-
terminal domain and the smaller one is an N-terminal domain (Radmark, 2003).
5-Lipoxygease is an ATP, iron, calcium dependent enzyme (Radmark, 2003).
Two tryptophan residues (W75 and W201) have been identified that bind ATP-
analogs, and incorporation of these analogs inhibited ATP stimulation of 5-
lipoxygease (Zhang, 2000). 5-Lipoxygenase contains a non-heme iron in the
active site, coordinated by His-367, His-372, His-550 and the C-terminal lie.
Iron acts as an electron acceptor or donator during catalysis. The enzyme is
inactive if the iron is 2 + (RMmark, 2000). Ca2 + can strongly stimulate 5-
lipoxygease reactions in vitro, depending on the assay conditions. For purified 5-
lipoxygease, half-maximal activation was obtained in the presence of l-2pM
calcium, whereas 4-10 |iM Ca caused maximal activation of the enzyme
(Radmark, 2000). Ca2 + can activate 5-lipoxygenase by inducing membrane
association (Radmark, 2003). The N-terminal domain of 5-lipoxygenase is a
23
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
beta-barrel with the typical ligand-binding loops that have been shown to bind
Ca2 + to the membrane. It is now well accepted that translocation of 5-
lipoxygenase from the cytosol to the nuclear envelope is a critical component of
enzyme activation (Golden, 2001). However it is unclear whether cytosolic 5-
lipoxygenase binds to the nuclear membrane from the cytoplasmatic side after
redistribution; or, whether it enters the nucleus first to associate then with the
membrane from the nuclear side (Werz, 2002).
m
t c M
& 1 ftai #
Figure 2.4 Model of 5-lipoxygenase (Based on rabbit reticulocyte 15-
lipoxygenase and reproduced from Radmark, 2003).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
2.3 ALOX5 Gene Polymorphism and Atherosclerosis
The 5-lipoxygenase pathway leads to a leukotrienes-mediated
inflammatory circuit in the artery wall and affects the development and
progression of atherosclerosis (Zhao, 2004). The identification of genetic
variants in the ALOX5 gene has important implications for understanding the
mechanism of atherosclerosis and identifying of high risk candidates before
symptoms arise.
2.3.1 Genetic Polymorphisms in the ALOX5 Gene and Cancer Risk
Lipoxygenase metabolic pathways are emerging as key regulators of
cell proliferation and neo-angiogenesis (Romano, 2003). ALOX5 gene is found
overexpressed in cancer cells and human tumor cells. A considerable number of
studies have confirmed that 5-lipoxygease activity promotes cancer cell
proliferation and survival (reviewed in Romano, 2003). Avis et al found the
growth control of lung cancer by interruption of 5-lipoxygenase-mediated
growth factor signaling (Avis, 1996). In the human colon cancer, 5-lipoxygenase
is important in the biosynthesis leukotrienes (Romano, 2003). Two genetic
polymorphisms in ALOX gene which are implicated in decreased colon cancer
risk have been identified, including -1752G— *A and -1699G— >A, which are both
located in a negative regulatory region of the promoter (Goodman, 2004).
25
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
2.3.2 Single Nucleotide Polymorphisms (SNPs) and the ALOX5 Gene
Single nucleotide polymorphism (SNP) is the most common forms of
polymorphism. An average density on available sequence of one SNP is with an
estimated frequency of one for every 1,200-1,500bp (Venter, 2001). SNP
analysis of the human genome promises to provide insight into common disease
susceptibility because they may directly contribute to the risk of developing a
disease. For example, a SNP in the coding sequence may cause an amino acid
change; one change in an intron may influence splicing site (Goldsmith, 1983);
or another change in a promoter region may modify transcription factor binding,
and effect mRNA level (Drazen, 1999); or SNPs in 3 ’ UTR of a gene, may
directly or indirectly affect mRNA stability (Bensen, 2003). People with such a
SNP allele have a higher risk for that disease than do people without that SNP
allele (Majewski, 2003). Many recent studies are focused on SNP discovery, but
there is a great number of SNPs associated with no physiological or functional
significance. The high frequency of SNPs that occur in the human genome
suggests that they can be used as markers for gene mapping studies (Kruglyak,
1997). The use of haplotypes containing multiple SNPs will increase the power to
detect linkage disequilibrium. Analysis of linkage disequilibrium allows
selection of informative subset of SNPs for genomic regions of interest. The
association between SNPs and a disease may indicate there is a possibility of
related genes in this region that contribute to the disease (Ott, 1997). So far 122
26
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
SNPs were identified in the AL0X5 gene: 12 in the promoter region, 5 in the
exons, and 11 in the 3’UTR. (http://snpper.chip.org/bio/viewsnpset/cxme945423/
T, 2004).
2.3.3 Gene Expression of the ALOX5 Gene
2.3.3.1 Gene Expression
Control of gene expression is achieved at four broad levels:
transcriptional control, post-transcriptional regulation and epigenetic
mechanisms and long range control of gene expression. Transcriptional control
of many genes is mediated by binding of diverse transcription factors to cis-
acting DNA motifs located in the DNA such as promoters, enhancers and
silencers. Post-transcriptional regulation of gene expression includes RNA
processing, mRNA transport, translation, mRNA stability, post-translational
modifications and protein degradation. Epigenetic effects on gene expression are
genetic phenomena without any DNA nucleotide change that affect gene
expression including DNA methylation, allelic exclusion and long range control
by chromatin structure (reviewed by Strachan, 2004).
2.3.3.2 Promoter Region and Gene Transcription
To understand the mechanism of transcriptional regulation, it is essential
to identify and characterize the promoter of a gene. The promoter is usually
27
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
located just proximal to or overlapping the transcription initiation site and
contains several sequence motifs with which transcription factors interact in a
sequence-specific manner. Promoter region is required for accurate and efficient
transcription initiation (Wasylyk, 1987). It can be divided into several
components: the core promoter, proximal promoter region, and distal promoter
regions (Strachan, 2004) as shown in Figure 2.5.
- ,1 I,. J
m - m * S 3
r M j i f t l i
Figure 2.5 Conserved locations in complex eukaryotes for regulatory
promoter elements bound by ubiquitous transcription factors (Reproduced
from Strachan, 2004)
The core promoter is generally located within -40 to +40 of the
transcription start site, and is recognized by the basal RNA polymerase II
transcriptional machinery (Kadonaga, 2002).There are several sequence motifs
which are commonly found in core promoters: TATA box, TFIIB recognition
28
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
element (BRE), and downstream core promoter element (DPE) (Butler, 2002).
The TATA box, a highly conserved A-T-rich region, is an element that directly
binds the transcription machinery to initiate mRNA synthesis. In humans,
approximately 32% of 1031 potential promoter regions contained a TATA box
motif (Suzuki, 2001). The BRE is a TFIIB binding site that is located
immediately upstream of some TATA boxes. TFIIB is able to bind directly to the
BRE in a sequence-specific manner. In vitro transcription experiments revealed
that the BRE helps the incorporation of TFIIB into a productive transcription
initiation complex (Lagrange, 1998). Some promoters contain neither TATA box
nor other alternative promoter elements. In those genes, transcription is often
initiated at multiple sites over a defined region (Hawkins, 1996). The DPE is a
sequence that is normally found (+28 to +32) downstream position and most
commonly found in TATA-less promoters (Kutach, 2000). TFIID binds
cooperatively to the DPE motifs. Core promoter activity is strongly reduced upon
mutation of DPE motifs in DPE-containing promoters (Burke 1996).
Enhancer and silencer elements contain binding sites for sequence-specific
transcription activators and repressors. They usually locate the upstream from the
core promoter, and play key roles in controlling the site and transcription levels.
Nucleotide mutations in upstream promoter regions may cause variations in gene
expression levels and be involve in disease predisposition (Hawkins, 1996).
29
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
2.3.33 Promoter Region of the ALOX5 Gene
The ALOX5 gene promoter was cloned (GENB ANK#M3 8191) and
characterized (Funk, 1989). There is no TATA box. It is G+C rich, has multiple
GGGCGG sequences and has multiple transcriptional start sites. The -179 to -56
DNA region appears to be essential for transcription initiation (Silverman, 2002;
Hoshiko, 1990). There are two positive regulatory regions between -3,700 and -
5,900 and between -854 and -931 from ATG translation start site, and two
negative regulatory regions between -1,557 and -3,400, and between -292 and
-727 as determined by deletion analysis of the promoter region (Hoshiko, 1990;
In, 1997). A c-Myb, NF-kB and AP-2 (Figure 2.6) consensus sites for
transcription factors have been examined (Hoshiko, 1990). Recently, analysis of
the promoter region of the ALOX5 gene suggested that SP1-binding motifs may
play an important role in gene transcription and drug response to inhibitors (In,
1997; Drazen, 1999). Dwyer reported that the mean intima-media thickness was
significantly increased in the patient group with two variant alleles in SP1
binding motif of ALOX5 gene, compared with carriers of the common allele
after being adjusted for age, sex, height, and racial factors (Dwyer, 2004). These
observations provided a reason to consider that genetic polymorphisms in this
region play a potential role in modifying ALOX5 gene transcription and mRNA
translation (In, 1997).
3 0
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
-1672 bp: c-myb consensus sequence
-1521 bp: RZR-response element
-893 bp and -1007 bp: SP1 consensus sequences
-443
N F -/C B IG F '( 3
-393
-343
IG F '( 3 A P-2
-293
-243
T O X D D G X D S fG Q O S A G G IO C D a C D IA G IO a S O G D D o C G K S W S O G S -193
S P 1 S P 1 S P 1 S P 1 S P 1
-143
A P-2
AGQa9GG|ll^ -93
A X A 3]G G IG G 3A 3G A Q X lG a330^^ -43
5fU IR -1 + 1
M e t
C C IlA Z A D 9 3 ]O O D G IG Q 0 C A C IQ X A G D C A G IG G n 0 3 C D 3 3 C A C rC ® D 3
A P IA 3ffC IA aG IC A 3C X 3aiD 3G G ^^
E xon 1
Figure 2.6 Promoter region of the ALOX5 gene (Sequence was copied
from http://www.ncbi.nlm.nih.gov)
2.3.4 Hypothesis
At steady state, AL0X5 mRNA is barely detectable in many cell lines,
but mRNA levels are increased following cell activation (Ring, 1996). Increased
expression of AL0X5 mRNA in leukocytes of atherosclerotic lesions compared
to normal subjects suggests an important role for transcriptional activation in the
pathogenesis of atherosclerosis (Spanbroek, 2003). Regulation of specific gene
31
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
expression depends on the interaction between trans-acting factors and the cis-
elements on the gene. Sequence variants in the 5’ flanking region of the ALOX5
gene may modify the interaction of regulatory proteins with the cis-elements,
and hence affect the amount of ALOX5 protein produced. If increasing the
amount of the ALOX5 protein produced, it would lead to increased biosynthesis
of leukotrienes which promote atherosclerosis. Sequence variants in coding
region could modify the protein derived from translation of ALOX5 mRNA. To
test this hypothesis, exons 1, 12 and 13, as well as 1670 bp 5’ flanking region of
the ALOX5 gene were scanned for mutations.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3.0 MATERIALS AND METHODS
3.1 Subjects and DNA Isolation
A total of 86 unrelated individuals were analyzed in sequence variants of
ALOX5 gene. Genomic DNA samples were isolated from freshly drawn blood
from 86 individuals by using phenol extraction by Nancy Chen (technician in Dr.
Reichardt’s lab).
3.2 PCR Amplification of ALOX5
Polymerase chain reactions (PCR) was used to selectively amplify the
1670 bp promoter region, 5’UTR + exon 1, exons 12 and 13 from genomic DNA.
Sequencing of exon 2 to exon 11 and exon 14 + 3’UTR have been done by
Nancy Chen (technician in Dr. Reichardt’s lab).
3.2.1 Primer Design
A web based software package called Primer3
(http://www.basic.nwu.edu/biotools/Primer3.html, 2004) was used in designing
primers for the amplification of the promoter region and exons 1, 12 and 13. The
sets of primers (Invitrogen, Carlsbad, CA) flank promoter region and exons 1, 12
and 13 respectively as shown in Figure 3.1, where the sequence of the template
is also shown. The primer annealing sequences for PCR are shown as bold and
underlined.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
c o c a a ia a c o s s s c m ttg a a a a a 3 & A A G R ftc a m s
TOGGB3GMT AACATITKX: MTTTCAAAA CFIACIAIAG C ?O 303G m
A T C A A G C A G T CTGGOOGT M AG30GIA C A ITA C A G A T CSGIGGSCm
GaKiraATGr q c a g a a k e a a p c a jr r im i t ia ia a t c a a . T r a c m r iA
im m sT G K : a s g a c a a c g c a a iq q g a a a a g a a e a a ig a a t t g a a c a m t
G A iG C KrQ G A c a a c d g s x a . t o g a c a k x a ACAeAATG AA t t t g a a 2 t c t
TXICfflDSCIC CAOXAIAAA AACIAACKA. A A A T G 3G T C A 033ATGIAM.
T G A A A A G C IA M f f l f f l A A T C C IA G A G G A A A A C C T A G GCIAAATCT
TTAAOflErr OTPSM3QGA. GTGGITTCrC agaiaggacc ccaaakicac
AAGOSOffiA A G A A A T T G G A CTTAAAGnA A A1A CTTTTG W XTTC AAAC
a tc a tc a a g a a a g k a a a a c a c a a o c o x a G M im im p m sG iciG i:
a a g k a ig ia todgkttaga s o r c m r c caq® jk ea t aaa iaaigca
ATICAAT®ff A A A A A A G A IA AMA30CCAG TITTO C A A A G M3IOW3CM'
ctgaataiac a ic ic k ia a m j b j o g atakxaaca aqcktgigaa
A A G A 3G T T C A AAG3CA3TIG (XAQ3JGCAC A A A C X X A A G A ®GEATS¥33
AGKKXTACA GQGACTCKX TQCTTCA3G ACAPGAA3X TIG3TGAGAA
T C T A Q 3 G A G C 03CCITTQGG GACTKACAT C0003003CC 03CGCA03G
TGRQCDCTG TnAAACITA Q O O SA G A IC A AIACA0903\ CrG TG TOOCC
CTCAfflOCET GQ3CIQ0CQ3 03033X 036 A3AQ33G333 QXA3GAGTG
G G D 3G G A A 06 TQ 33G G TC A G aXXXAQOOG CGGSAQXG C OCAG®GCG
0333AAA03T TCTXX30CC CTKXA33CA TITGCXXBCC GOGAITCA0\
GAGC03OX GK3CCXCIG GCCKXXCTA GACAG03333 CAIUKXAGI
T G IG O O G T O G 01X 300010 Q33J3CCPC TQ3COHOC T G Q Q C C T G Q 3
CQ333ICKG GOXXCGCCr GOCDOXXEA G G A Q 303C A G GKXAGXAG
IG AATAAGaC C G 33CTCA A G GAGCCICIET GCTCCAfflAT OCAICCICAG
T A T C A Q O G G T G G S G T G G C C T C C IX C A G C A A Q X C IT C rei TTCIC1XATC
OXOQOTCTT OXOIGGAGA CID003GAQC AOOOOIOOIC (3AGIAC03C
AAGIG 3GACT ©fflACTIGG GfflSCCAGA QGCIGrGOCr A3XCTIGIAG
GGAGI00003 CAGCIXCAX 03AG3GCCIA C A Q G A G C C T G GD C TTG 3G D 3
A A G C X E A G 3 C A G 33A G Q C A E G33AAAQ33T OSWOAATT G A Q G A G A G A A
c m s rs ff io s aa3 g ® i ® g aoroGCAGc OG AQ snax: ooagkxxxt
G3CIGCA33A A C A G A C A C C T 03CIGAG3A3 M T O G a . G33ffiX XX;
t o o x x g o x gagsosaggt a x m r a s r a o a m x o t g a a s g b s s
G S G SG R A G E A CP333333X Q 3G G 33G G G G Q03G3GG333 G3G033Q3GC
A G C 03G G A Q C C K 33A G O C A G AC0333Q33G GGC033GA CC Q 3G G C C A G G 3
A0CAGIG3IG G G A G G A Q 3 0 T G C G Q O G C E A G A3G033ACAC C IG S m X C
G03C03SGQC TCXXGGOOr O3C3GCI0GC (XXXOTXXG C E K D G D C C T C
(i) PCR primer annealing sites on the promoter region between -1803 to -180 bp
relative to ATG (The sequence in grey box is translation start site ATG as +1.)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
P 0 3 3 3 3 S Q Z C D 3 3 g X a3 ACGIGCQa C G3XQ3GKCC GG3G DCSG3S
XXSGT3GIG QfflGGB33CT Q3Q3CQ3IEG AT303G?OC CTQGWXSjC
axxoJSGc; ia5XxW T,crrrr^ro qH ^ax^^a^toaxar:
C m X G G IC i«XSD 33C !C A ( 3B 3 S3 &X& GEQQEKGX Q 3 3 CK»CG
k m o a o o K S G xn r; G ra a x ro a e aaaaaoocftG o® gaaqcrc
C T a c n a a c A j y r o c r a c m a ^ a a c r a c Q f f G U H E GGGTfflQooc
GG3033333S. C033IQ3SG: Q3333Cn®3 9103310335 gjOXOSETT
G G B O G
(ii) PCR primer annealing sites on 5’UTR and exon 1 of AL0X5 (The sequence
in grey box is 5’UTR and exon 1 is shown as bold.)
G Q G ftM C D Q S (30030351X3 Q33G33Q3X
03K3GQ3D33 T C 2G 93A C T G GGOOOCCC 030CG G IG G T T O C A 003E M 3
G CO TX CEA A G K 33S T C A A G 2Q XX33& G C A3C3t3I033A. G IW C JX2C C
GD3SH30CT TC3033COO 033X »X A C Q 3 0 3 C G 5 [D C & A 3 C T C Q S O C A
G 3IA 3G C R G G G O C n S S C D O G CK3333»Q3S CIOXTICIC AM9033CIG
c r a a i r c m : a x o x o s c T c r o c a a a c s r i o x r w r c r
(iii) PCR primer annealing sites on exon 12 of AL0X5 (exon 12 is shown as
bold.)
03333RftftGft. Q G K T Q G S C Q S J O G C T O G S G C C D 3 C T O 3 M 3 T T 3 Q 3 G Q 3 C A C
GG3GAG©03 G Q G O X M 33G GQ3G3FG9S C SQ C M 393Cr T 099G G G T Q 3
COOdCrEQC TQ3C X53I03T COT333BG EV 0530103105 TO3IQGMDC
Q3&MOCOX OCCAMCMG 03*3333002 C&0CE2CI03 O M 3333IG
G D a a S M S £G3AGMQ3T 0 3 0 0 3 X 3 3 Q333A03333 Q3033K351G
CE333VKXD3 G3QC8GIGT 0 3 3 3 3 3 3 0 0 O C K G riQ G ftG G&AAAQ0G:
'K 5W 3C 3G Q G 0 0 0 3 3 9 3 3 3 CACZffiXOV 0 9 1 0 0 3 3 0 G3HAM303G
TT O caraox araG soarar g
(iv) PCR primer annealing sites on exon 13 of AL0X5 (exon 13 is shown as
bold.)
Figure 3.1 PCR primer annealing sites on promoter region, exons 1,12 and
13 of the ALOX5 (Sequence was copied from http://www.ncbi.nlm.nih.gov. The
primer annealing sequences for PCR are shown as bold and underlined.)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3.2.2 Polymerase Chain Reaction (PCR)
The lOx PCR buffer, Taq DNA polymerase were from Invitrogen
(Invitrogen, Carlsbad, CA). The dNTP mixture was from Takara Bio.INC. (Otsu,
Japan). The PCR reagents were optimized to obtain required PCR products as
shown in the Table 3.1. Amplification of the highly G+C rich promoter region
required an additional 2.5% (v/v) of DMSO.
PCR reaction was carried out in a Peltier Thermal Cycler (MJ Research,
MA) as follows: an initial denaturation at 95 °C for 15 min, 35 cycles of
denaturing, annealing and extension for 30 seconds at 95 °C; 30 seconds at
50-70 °C and 1-2 min at 72 °C and a final extension for 7 min at 72 °C.
Table 3.1 PCR reagents, volumes and final concentration
Reagent Volume
Final
Concentration
PCR buffer (lOx) withl5mM MgCl2
dNTP (2.5mM) mixture
Forward-primer (lOpM)
Reverse-primer (1 OpM)
Enzyme TAQ (5U/pL)
Genomic DNA
DMSO (100%)
Total (add deionized water)
5 pi
4 pi
2 pi
2 pi
0.4pl
1-2 pi
0-1,25pl
50pl
lx
0.2mM each
0.4pM
0.4pM
2.0U/reaction
10-20ng
0-2.5%
* lx PCR buffer: 20mM Tris-HCl (pH8.4), 50mM KC1, 1.5mM MgCl2.
36
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3.3 Gel Electrophoresis and PCR Product Purification
PCR product was electrophoresed on lx Tris-Acetate-EDTA (TAE) buffer,
1.5% agarose gel to confirm the size and amount.
The remaining PCR volume (48pl) was loaded on lx TAE buffer, 2-4%
agarose gel, and DNA was extracted by the using QIAquick PCR gel extraction
kit (Qiagen, Valencia, CA).
After the purification, 2pi of purified DNA samples were loaded on the
1.5% agarose gel to check column recovery.
3.4 Automated DNA Sequencing
Sequencing reaction were carried out in a 10 pi containing 3.2 pmol
primer, 15-50ng purified PCR products, 2-4pl of ABI Prism Terminator Big Dye
Cycle Sequencing Ready Reaction Mix and lx buffer (5x: 400mM Tris-Hcl, pH
9.0 and lOmM Mgch) lpl and dHaO. As shown as Figure 3.1, the primers
previously described were used. However, due to the length of the promoter
region PCR products (1670bp), internal primers also were utilized as shown in
Table 3.2.
The cycle sequencing reaction was carried out in a Peltier Thermal
Cycler (PTC-100™, MJ Research, Inc., Waltham, MA) as follows: an initial
denaturation at 98 °C for 3 min, 30 cycles of denaturing, annealing and extension
for 30 seconds at 98 °C, 15 seconds at 50°C and 4 min at 60°C, respectively.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 3.2 Internal primers within the promoter region used during
automated sequencing _____ _
Ki:s»i,r: o i .ntwi'ist 'esi-sC prim e; (5’!o i') dnti-tcns. prim er to 3’j
* - 1803_-1400 A A A G A A C A G C G T T G G T G G A T T A A A C C A C T G C C T A C A A T A A C A
* -1 4 0 0 _ - 1 0 0 0 C C C A A A A T C A C A A G C G A C A A A C C C A A A G G C G G C T G C C T A C A T T
* -1 0 0 0 _ - 6 0 0 C A C G C A C G G T G A G C T A C T G T T G C T T C T T C A C T G G C T G G A C C T
* -6 0 0 _ - 1 8 0 C A G A A T C C A T C C T C A G T A T C A A C C T C G C C T C G G G C G G G G C A G
♦Position is reference to the ATG translation start site as +1.
After sequencing, the solution was purified with size exclusion Autoseq
G-50 columns (Amersham Biosciences, Piscataway, NJ) to remove the
unincorporated fluorescently labeled ddNTPs, and then dried by placing inside a
DNA Speed Vac (Savant Instruments, Farmingdale, NY) for 15 min. The pellet
was resuspended in 10 pi of Hi-Di formamide (Applied Biosystems, Foster City,
CA) and was loaded into a 96-well plate. The solution was heat denatured at
98°C for 15 min and then iced at least 5 min. The 96-well plate was loaded onto
the ABI Prism 3100 DNA Sequencer. After termination of electrophoresis,
nucleotide sequences were analyzed with ABI Sequence analysis software (Seq
Scape version 1.1).
38
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
4 RESULTS
4.1 Polymerase Chain Reaction (PCR) Results
The PCR conditions were optimized to obtain a strong and specific
amplification band. The PCR product is shown on a 1.5 % agarose gel in Figure
4.1.
1600bp
■ ■ M P I
m
(i) PCR product of the promoter region Negative control
M
356bp
W
tfcii ■ B L
Negative control
(ii) PCR product of 5’UTR + exon 1 (there is one sample that did not
amplify).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(iii) PCR product of exon 12
Negative control
M
(iv) PCR product of exon 13
Negative control
Figure 4.1 PCR products (M is 1Kb plus DNA ladder (Invitrogen, Carlsbad,
CA). The bands are PCR products of different samples.)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
4.2 Purified PCR Products
After purification by using QIAquick PCR gel extraction kit (Qiagen),
purified PCR product was loaded on the 1.5% agarose gel to confirm the quality
of the template. The result was shown as a band on the agarose gel in Figure 4.2
M
1600bp
(i) Purified PCR product of promoter region
356bp
IH L
(ii) Purified PCR product of 5’UTR + exon 1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(iii) Purified PCR product of exon 12
M
370bp —►
(iv) Purified PCR product of exon 13
Figure 4.2 Purified PCR products (M is 1Kb plus DNA ladder (Invitrogen,
Carlsbad, CA). The bands are purified PCR product from different samples.)
4.3 Mutation Identification
To avoid the false result in screening sequence variants from the ALOX5
gene, I confirmed the mutations by sequencing in both orientations except two
SNPs: T with A at position -1744, G with A at position -1753 relative to ATG
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
translation start site. The reason is that the positions of these two SNPs are close
the beginning of the PCR fragment. The ABI 3100 automatic sequencer can not
detect the first 50 bp from the DNA template. Fourteen sequences of sequence
variants for the promoter region, 5’UTR, exons 1, 12, and 13 were shown
together in Figure 4.3, 4.4, 4.5.
(i) Wild type and mutant sequences of type -1753 G— »A (Reverse strand)
Wild ty p e-1753 C-+T
Mutant -1753 C-+T
Wild type -1744 A— > T Mutant -1744 A— > T
m S f- A " • * G .G '
(ii) Wild type and mutant sequences of -1744 A— * T (Reverse strand)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Wild type -1700 G— »A
Mutant -1700 G— >A
. 43 ' J. r- ? A '-7 . • /j < 1 - ’ "VC * ' *
(iii) Wild type and mutant sequences o f -1700 G— >A
Wild type (-1366) to (-1361)
GTTAAA
Deletion (-1366) to (-1361)
GTTAAA
'B M m m M R
(iv) Wild type and deletion of (-1366) to (-1361) GTTAAA (The deletion reflects
the overlapping of two alleles in this heterozygote. The deletion can be
confirmed by sequencing the reverse strand of DNA)
44
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Wild ty p e-1145 G— > C
Mutant -1145 G— ► C
v v . * < u • ‘ -r W H
' ' 2 t i . 23S
■ ■ ^ » * ! . . ■ ■ ■ " !
£ /,
£
t
*
(v) Wild type and mutant sequences o f -1145 G— ► C
Wild type -839 G— >A Mutant -839 G— >A
xi c;
• 2 0
f-*1 : fi .*
130
(vi) Wild type and mutant sequences of -839 G— >A
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Wild type -754 C-+G Mutant -754 C— >G
(vii) Wild type and mutant sequences of -754 C— >G
Wild type -557 T-+C Mutant -557 T->C
£ ‘ ' • 2 - i ' C -* '
- 1 iv.r. r
(viii) Wild type and mutant sequences of -557 T— >C
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Wild type -310 A— >G Mutant -310 A— >G
m m m k > > .
"f.
1
LJul..
(ix) Wild type and mutant sequences of -310 A— >G
Wild type -59 C— >T Mutant -59 C— »T
(x) Wild type and mutant sequences of -59 C— >T
Figure 4.3 Sequence variants were found in promoter region of ALOX5
(Position is in reference to the upstream ATG translation start site).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Wild type -28 G->C
Mutant -28 G— > C
20
»
5 * 1
(i) Wild type and mutant sequences of -28 G— »C
Wild type -15 C— » G Mutant -15 C-» G
m g : m C < < 4
(ii) Wild type and mutant sequences o f -15 C— » G
Figure 4.4 Sequence variants were found in 5’UTR of ALOX5 (Position is in
reference to the upstream ATG translation start site).
48
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Wild type 21 C-*T Mutant 21 C-+T
• ' ' i C ? ’ 'A
a . . 129 .
Vi V ’ G " 5 C
(i) Wild type and mutant sequences of 21 C— >T
Wild type 1728 A-»G
(AA)
O ' * G ' .<
Mutantl728 A— *G
(AG)
• S ' ; :e A\y
Mutant 1728 A— »G
(GG)
f .
; r
I- „ U A
(ii) Wild type and mutant sequences of 1728 A— »G
Figure 4.5 Sequence variants were found in exons 1 and 13 of ALOX5.
(Position is in reference to the cDNA of human ALOX5 gene.)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
4.4 Data Analysis
4.4.1 Hardy Weinberg Equilibrium
The Hardy Weinberg equilibrium states that, under certain conditions,
after one generation of random mating, the genotype frequencies at a single gene
will become fixed at a particular equilibrium value. In the simplest case of a
single locus with two alleles A and a with allele frequencies of p and q,
respectively, the Hardy Weinberg equilibrium predicts that the genotypic
frequencies for the AA homozygote to be p 2, the Aa heterozygote to be 2pq and
the other aa homozygote to be q2 (Merten, 1992).
Hardy-Weinberg equilibrium as a method to identify and reduce
genotyping errors generated as a result of the genotyping process itself in
population-based studies (Gordon, 2001). I did the Hardy Weinberg equilibrium
calculation for my results. Table 4.1 shows the distribution of the alleles and the
frequencies of SNPs from promote region, exons 1, 12 and 13 of the ALOX5
gene. Expected number and x 2 test with one degree of freedom for two alleles
were performed, and results are also shown in Table 4.1.
For -1753G— >A, -1700G-+A, (-1366) to (-1361) deletion, -839G ^A and
-557T— »C:
p= observed wild type alleles/ total alleles = (2*56+30) / (2*86) = 0.825
q= observed mutant alleles/ total alleles = 30/ (2*86) = 0.174
Expected wild type homozygote number = n*p2=86*0.825*0.825 = 58.6
50
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Expected heterozygote number = n*2*p*q = 86*2*0.825*0.174 = 24.8
Expected mutant homozygote number = n* q2 = 86*0.174*0.174 = 2.6
x2(d f=1 ) = [(56-58.6)758.6]+ [(30-24.8)2 /24.8]+ [(0-2.6)2 /2.6] = 0.1+1.09+2.6
= 3.8 (P>0.05)
Thus, these alleles distribution in population confirms well to the Hardy-
Weinberg equilibrium (P>0.05).
For -59C— >T , -28G ^C , -1 5 C ^Q 21C-+T:
p = observed wild type alleles/ total alleles = (2*59+27) / (2*86) = 0.843
q = observed mutant alleles/ total alleles = 27/ (2*86) = 0.157
Expected wild type homozygote number = n*p2 =86*0.843*0.843 = 61
Expected heterozygote number = n*2*p*q = 86*2*0.843*0.157 = 23
Expected mutant homozygote number = n* q2 =86*0.157*0.157 = 2
x2(df=i) = [(59-61)761] + [(27-23)723] + [(0-2)72] = 0.05+0.65+2 = 2.7
(P>0.10)
Thus, these alleles distribution in population confirms well to the Hardy-
Weinberg equilibrium (P>0.10).
For -754C— >G:
P = observed C alleles/ total alleles = (2*76+10) / (2*86) = 0.94
q= observed G alleles/ total alleles =10/ (2*86) = 0.06
Expected CC number = n*p2 =86*0.94*0.94 = 76
Expected CG number = n*2*p*q=86*2*0.94*0.06 = 9.7
51
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Expected GG number = n* q2 = 86*0.06*0.06 = 0.3
x2(df=i ) = [(76-76)2 /76] + [(10-9.7)2 /9.7] + [(0-0.3)2 /0.3] =0.3 (P>0.10)
Thus, these alleles distribution in population confirms well to the Hardy-
Weinberg equilibrium (P>0.10).
For 1728A— »G:
p = observed A alleles/ total alleles = (2*71+14) / (2*86) = 0.91
q = observed G alleles/ total alleles=14+2/ (2*86) = 0.09
Expected AA number = n*p2 = 86*0.91*0.91= 71.2
Expected AG number = n*2*p*q = 86*2*0.91*0.09 =14.1
Expected GG number = n* q2 = 86*0.09*0.09 = 0.7
x2(df=i )= [(71-71.2)771.2] + [(14-14.1)714.1] + [(1-0.7)70.7] = 0.2 (P>0.10)
Thus, these alleles distribution in population confirms well to the Hardy-
Weinberg equilibrium (P>0.10). The frequencies of the -1744T— >A, -1145G-^C
and -310A— »G are too low (less than 2%), hence further analysis was not
performed.
4.4.2 Haplotype Analysis of ALOX5 Gene
Haplotypes are sets of alleles on the same small chromosomal segment,
and they tend to be inherited together as a block (Strachan, 2004). The use of
haplotype analysis provides an accurate method to assess the relationship
between variation in a defined genetic region and a disease (Fallin, 2001). It also
52
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
can increase power in situations where combinations of polymorphisms affect
gene function (Terry, 2000). 27 sequence variants which frequency was more
than 5% found in the ALOX5 gene from 86 individuals were used for hyplotype
analysis. Table 4.2 shows their position and frequency. Part of data was from
Nancy Chen (technician in Dr. Juergen Reichardt’s lab) as shown in grey box in
Table 4.2.
Haplotypes of the ALOX5 gene were analyzed by using PHASE, version
2.0 (Stephens, 2003). There are 12 haplotypes in the ALOX5 gene showing
frequencies of more than 2% among the haplotypes observed (Table 4.3). They
covered 67.3% of all haplotypes in the population.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 4.1 H ardy Weinberg equilibrium (p values are calculated from the Chi-
Square test)
Position Allele Observed Expected Frequencies
P
Value
GG 56 58.6 0.65
a -1753G— »A Promoter GA 30 24.8 0.35 >0.05
AA 0 2.6 0
TT 85 83.9 0.99
a -1744T— > A Promoter TA 1 2 0.01 >0.05
AA 0 0.1 0
GG 56 58.6 0.65
H -1700G— > A Promoter GA 30 24.8 0.35 >0.05
AA 0 2.6 0
m GTTAAA 2*(GTTAAA) 56 58.6 0.65
(-1366) to (-1361) Promoter GTTAAA 30 24.8 0.35 >0.05
deletion
. . .
0 2.6 0
GG 85 83.9 0.99
a -1145G— > C Promoter GC 1 2 0.01 >0.05
CC 0 0.1 0
GG 56 58.6 0.65
m -839G— > A Promoter GA 30 24.8 0.35 >0.05
AA 0 2.6 0
CC 76 76 0.88
■ -754C—G Promoter CG 10 9.7 0.12 >0.05
GG 0 0.3 0
TT 56 58.6 0.65
m -557T— > C Promoter TC 30 24.8 0.35 >0.05
CC 0 2.6 0
AA 85 83.9 0.99
m -310A— »G Promoter AG 1 2 0.01 >0.05
GG 0 0.1 0
CC 59 61 0.69
m -59C— > T Promoter CT 27 23 0.31 >0.05
TT 0 2 0
GG 59 61 0.69
m -28G— > C 5’UTR GC 27 23 0.31 >0.05
CC 0 2 0
CC 59 61 0.69
■ -15C— *G 5’UTR CG 27 23 0.31 >0.05
GG 0 2 0
CC 59 61 0.69
*21C-»T Exon 1 CT 27 23 0.31 >0.05
TT 0 2 0
AA 71 71.2 0.83
*1728A— > G Exon 13 AG 14 14.1 0.16 >0.05
GG 1 0.7 0.01
a Position is in reference to the ATG translation start site (+1) of human ALOX5
gene
^Position is in reference to the cDNA of human ALOX5 gene
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 4.2 Position and frequency of polymorphisms used in hyplotype
analysis______ _________ _______________ __________ ______ _____
# M u tan t Position
H eterozygote
Frequency
# M u tant Position
Heterozygote
Frequency
1 -1753G — >A Promoter 35% 2 -170QG— >A Prom oter 35%
3
GTTAAA
deletion
Promoter 35% 4 -839G— >A Prom oter 35%
5 -754C— »G Promoter 9.7% 6
-557T— >C
Prom oter 35%
7
;3-m
it ilii it tr ti;
Promoter 51% 8 -59C-+T Prom oter 31%
9 -28G— >C 5 ’UTR 31% 10 -15C— »G 5 ’UTR 31%
11 *21C— »T Exon 1 31% 12 802 f t > i Intron 1 31%
13 * - \ Exon 2 29% 14 4 Iti- *A Intron 2 10%
15
1 16 3_( . \ Intron 4 7% 16 1 I6 -5 ( i--*-a Intron 4 7%
17 ’ " 6 0 G -A Exon 6 7% 18 3-H3G- 'A Intron 6 19%
19 — U Intron 7 22% 20 6 : 7( -*l Intron 8 5%
21 6 4 1 \ — !-i Intron 8 5% 22 13t i- -* A Intron 10 5%
23 J 2 f - 1 Intron 10 22% 24 5 1 I * ■ ( Intron 11 23%
25 *1728A— >G Exon 13 16% 26 D e.ti-m -\t 3 ’UTR 7%
27
m m
3 ’UTR 8%
Position of # 1 to #10 mutants is in reference to the ATG translation start site
(+1) of human ALOX5 gene.
^Position is in reference to the cDNA of human ALOX5 gene.
Position of #12, #14, #15, #16 and #18 to #24 mutants is in reference to their
individual introns.
The reference sequences were from http://www.ncbi.nlm.nih.gov.
55
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 4.3 Haplotype frequencies of ALOX5 gene (Haplotypes which is a
frequency more than 2% are listed).
Chromosome 10 ALOX5 Gene Region
Frequency
(%)
#1 #2 #3 #4 #5......................................................................................................#25 #26 #27
H tl (G-G-(GTTAAA)-G-C-T-5(GGGCGG)-C-G-C-C-C-G-G-G-G-G-A-C-A-C~G-C-C-A-AC-C) 28.23
Ht2 (G-G-(GTTAAA) -G-C-T-S(GGGCGG)-C-G-C-C-C-G-G-G-G-G-G-T-A-C-G-C-T-A-AC-C) 5.91
Ht3(G-G-(GTTAAA)-G-C-T-6(GGGCGG)-C-G-C-C-C-G-G-G-G-G-A-C-A-C-G-C-C-A-AC-C) 5.13
Ht4 (G-G-(GTTAAA) -G-C-T-3(GGGCGG)-C-G-C-C-C-A-G-G-G-G-A-C-A-C-G-C-C-A-AC-C) 4.47
Ht5 (A-A- (deletion) -A-C-C-4(GGGCGG)-T-C-G-T-T-G-G-G-G-G-A-C-A-C-G-C-C-A-AC-C) 4.21
Ht6 (G-G- (deletion) -G-C-T-5(GGGCGG)-C-G-C-C-C-G-G-G-G-G-A-C-A-C-G-C-T-A-AC-C) 3.88
Ht7 (G-G- (deletion) -G-C-T-5(GGGCGG)-C-G-C-C-C-G-G-G-G-G-A-C-A-C-G-T-C-A-AC-C) 3.43
Ht8 (A-A- (deletion) -A-C-C-4(GGGCGG)-T-C-G-T-T-G-G-G-G-G-A-C-A-C-G-T-C-A-AC-C) 3.38
Ht9 (G-G-(GTTAAA)-G-C-T-5(GGGCGG-C)-G-C-C-C-G-G-G-G-G-A-C-A-C-G-C-C-G-AC-C) 3.03
HtlO (G-G-(GTTAAA)-G-G-T-S(GGGCGG)-C-G-C-C-C-A-A-G-G-G-A-C-A-T-G-T-C-A-del-C) 2.87
H tll (A-A- (deletion) -A-C-C-4(GGGCGG)-T-C-G-T-T-G-G-A-G-G-A-C-A-C-G-C-C-A-AC-C) 2.82
H tl2 (G-G-(GTTAAA)-G-C-T-3(GGGCGG)-C-G-C-C-C-A-G-G-A-G-A-C-A-C-G-C-C-A-AC-C) 2.80
The reference sequences were from http://www.ncbi.nlm.nih.gov.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
5.0 CONCLUSION AND DISCUSSION
5.1 Conclusion
Atherosclerosis affects one in four persons and causes 42% of all deaths
in the United States (American Heart Association, 2004). It is the primary cause
of coronary artery disease and stroke which are a major health concern.
Atherosclerosis is a complex disease involving many genetic and environmental
factors (Renke, 2003). Although a number of risk factors have been identified
which appear to influence the development of atherosclerosis, many people with
this disease lack relationship with the well-recognized risk factors such as
smoking, diet, high LDL cholesterol, hypertension and some genetic factors.
Present experience suggests that the relative reduction of atherosclerosis risk in
tightly managed patients in clinical trails of ranges from 20-40% (reviewed in
Renke, 2003). Therefore, research should address new risk factors that contribute
to the remaining 60-80% of atherosclerosis cases. Finding these risk factors will
help us identify disease mechanisms, develop new and specific the therapeutic
intervention, improve the presymptomatic diagnosis and prevention.
Recent studies have found that the ALOX5 gene involved in accelerating
atherosclerosis (Lusis, 2004). My investigations utilized PCR amplifying and
automated sequencing of the protein coding region exons 1, 12 and 13 and
promoter region of the ALOX5 gene. Genomic DNA samples were available
57
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
from 86 individuals. A total o f fourteen sequence variants were identified (Table
4.1). Seven sequence variants in the promoter region, two substitutions in the
5’UTR, two silent substitutions in exons 1 and 13 were found frequently. Other
three SNPs were seen only in one or two samples.
Four SNPs of these positions described above have been published so far:
-1700G— >A, -839G— >A in the promoter region (http://snpper.chip.org/bio/vie w-
snpset/), 21C— >T (In, 1997) in exon 1, 1728A— »G (http://www.ncbi.nlm. nih.
gov/SNP/) in exon 13. Among published data, one published SNP 126C— »A in
exon 1 was not found in this experiment, this maybe because small sample size
and low SNP frequency.
The importance of these sequence variants will be discussed below.
5.2 Promoter Analysis
Previous work has linked genetic polymorphisms o f a promoter region of
the ALOX5 gene with asthma. In 1999, Drazen et al. described a variable
number of tandem repeats (VNTR) in the promoter-region that result in a
decreased transcriptional activity. In patients with asthma, only those individuals
that expressed the wild-type ALOX5 gene promoter responded well to therapy
with a 5-lipoxygenase inhibitor (Drazen, 1999). Recent studies have found that
these VNTRs identify a subpopulation with increased atherosclerosis (Dwyer,
2004). The ALOX5 promoter has been characterized now. It has both positive
58
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
and negative regulatory regions, and contains numerous consensus binding sites
for many known transcription factors (Hoshiko, 1990). Thus, these observations
provide reason to believe that sequence variants in the promoter region of the
ALOX5 gene may modify gene transcription.
1670 nucleotides in promoter region of the human ALOX5 gene were
investigated. Nine nucleotide substitutions and a 6-base deletion were found.
These variants are: substituting C with T at position 59, A with G at position 310,
T with C at position 557, C with G at position 754, G with A at position 839, G
with C at position 1145, G with T at position 1700, T with A at position 1744, G
with A at position 1753 and a 6-base deletion at position 1366 (Table 4.1). Six
sequence variants located in negative regulatory region, and two variants located
in positive regulatory region of the ALOX5 gene. They may have an influence on
the expression level of a gene by different transcription factors recognizing. It
means that these sequence variants may lead to increased ALOX5 mRNA level
and hence increase in the 5-lipoxygenase enzymes. If more 5-lipoxygenase
enzymes are produced, then biosynthesis of leukotrienes will be increased. The
enzyme 5-lipoxygenase plays an important role in regulating the amount of
leukotrienes that are known as inflammatory mediators involved in development
and progression of atherosclerosis (Radmark, 2003), and earlier observation
support that atherosclerosis is an inflammatory process (Ross, 1999).
59
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
For each transcription factor binding sites that are either deleted or
generated by the nucleotide exchange are as shown in Table 5.1. The analysis is
based on “Databases on Transcriptional Regulation” (Heinemeyer, 1998,
http ://www. cbrc .jp/research/db/ TFSEARCH .html).
Table 5.1 TFSEARCH 2.0 search result
SNP
Wild type Mutation
T F
TF
-1744T— > A AML-1A/Oct-1 Ets
-1700G— »A - CDP
- 1145G— >C USF -
-59C— >T E47 -
-28G— >C - MZF1
5.3 Substitutions in the 5’ Untranslated Region (5’UTR)
Two substitutions (substituting C with G at position 15 and G with C at
position 28) were identified in the 5’UTR. The 5TJTR is known to play an
important role in the post-transcriptional regulation of gene expression via
different mechanisms (Pelletier, 1985). Firstly, context and structure of the
5' UTR significantly affect the initiation of translation. A comprehensive
60
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
computational analysis of 5 'UTR sequence in low-expression mRNAs and high-
expression mRNAs was carried out (Kochetov, 1998). Statistically, 5 'UTRs that
enable efficient translation are short, have a low GC content, are relatively
unstructured and contain favorable 5’UTR features that may help efficient
translation (Kochetov, 1998). Secondly, a cap-binding protein, eIF4 binds to the
7-methylguanosine cap at the 5’UTR. Without this cap, the binding of mRNA to
the ribosomal subunit is often not completed, and eIF4 is also critical for the
translation to proceed (Shatkin, 1985). Thirdly, 5’UTR usually contains a
response or binding element that is important for regulating translation. Mutation
of the 5’ UTR may affect the efficiency of mRNA translation (Morle, 1985).
Thus these two mutations in the 5 ’ UTR of ALOX5 gene may associate with
affecting translation initiation, translation efficiency (Flavio, 2002).
5.4 Silent Substitutions
Two silent substitutions were identified within the protein-coding region:
one is in codon 7 (Thr) and one is in codon 576 (Pro). Although some studies
revealed that SNPs in the coding regions of two human mRNAs can cause
different secondary structure folds o f mRNA (Shen, 1999), or lead to activation
of a splice site in protein coding region (Goldsmith, 1983), and thereby causing a
quantitative decrease in mRNA accumulation. However, a recent study showed
that these two SNPs in the ALOX5 gene were not located in the positions that
61
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
would have functional consequences in modifying the capacity to appropriately
splice intronic RNA (reviewed by In, 1997). In contrast to these two SNPs
without likely functional consequences, translation efficiency could be affected
by using a different codon (Milland, 1996). Thus these two SNPs identified in
the ALOX5 gene may have an effect on gene expression.
5.5 Future Work
In 2004, Dwyer et al. found that artery walls were as much as 18 percent
thicker among participants who did not have the normally 5 Spl binding motifs
in the promoter region of human ALOX5 gene (Dwyer, 2004). More sequence
variants in 5’ flanking region of the ALOX5 gene were found from my
investigations. They may play a role in atherosclerosis.
To clearly assess the effect of these sequence variants on gene expression
and their potential effect on atherosclerosis, in vitro reporter gene expression
analysis such as chloramphenicol acetyltransferase assay, luciferase assay, p-
galactosidase assay, secreted human placental alkaline phosphatase assay and
green fluorescent protein assay should be done. I describe here the luciferase
assay. Firstly, preparation of reporter constructs: luciferase reporter plasmid is
used in the reporter gene assay to examine the ALOX5 gene promoter activity.
The promoters can be obtained by PCR amplifying wild type and mutant type
DNA from genomic DNA isolated from individuals or using a site-directed
62
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
mutagenesis kit to obtain promoter containing the SNPs of interest from wild
type promoter. Restriction enzyme digestion sites can be introduced into the
amplified fragments. These fragments will be digested with Xhol, Kpnl or
Hindlll, and then ligated directly into digested promoterless plasmid vector.
Direct sequencing should be done to confirm the DNA sequence of interest.
Secondly, the recombinants will be transfected into a chosen mammalian cell
line, and then grown. Finally, luciferase activity can be measured in cell lysates
using the Luciferase Assay System. The amount of light produced is compared
with that of the wild type. If there is more luciferase activity, it means that
sequence variants in promoter region of the ALOX5 gene cause more gene
expression, and hence increase the amount of ALOX5 protein.
In addition, the sequence variants that were identified may be useful as
genetic markers for the identification of atherosclerosis risk. Dwyer et al. have
found the strong link between a variable number of tandem repeats (SP1 binding
motifs) in the promoter region of the ALOX5 gene and a risk for atherosclerosis
(Dwyer, 2004). In my investigation, nine sequence variants in 5’ flanking region
of the ALOX5 gene were found frequently. If in vitro reporter gene expression
analysis suggests some of these sequence variants can lead to increased ALOX5
gene expression. We need to further assess the association between these
sequence variants and risk of atherosclerosis in a case-control study.
63
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
REFERENCES:
Aiello RJ, Bourassa PA, Lindsey S, Weng W, Freeman A, Showell HJ;
Leukotriene B4 receptor antagonism reduces monocytic foam cells in mice;
Arterioscler Thromb Vase Biol 22: 443-449, 2002.
American Heart Association; Heart and Stroke Statistical Update; Dallas:
American Heart Association, 2004 Update.
American Heart Association; Heart and Stroke Statistical Update; Dallas:
American Heart Association, 2005 Update.
Anderson RN, Guyer B, Freedman MA et al; Annual summary of vital statistics:
trends in the health o f Americans during the 20th century; Pediatrics 106: 1307-
1317, 2000.
Avis IM, Jett M, Boyle T et al; Growth control of lung cancer by interruption of
5-lipoxygenase-mediated growth factor signaling; J Clin Invest 97(3): 806-813,
1996.
Ball K, Turner R; Smoking and the heart: The basis for action; Lancet 2(7884):
822-826, 1974.
Barrett CE, Bush TL; Estrogen and coronary heart disease in women; JAMA
265: 1861-1867, 1991.
Bennett RM; Reactive oxygen species and death; Circulation Research 88: 648-
652, 2001.
Bensen JT; Langefeld CD; Li L; Mccall CE; Cousart SL; Dryman BN; Freedman
BI; Bowden DW; Association of an IL-1A 3UTR polymorphism with end-stage
renal disease and IL-1 expression; Kidney International 63(4): 1211-1219, 2003.
Berliner JA, Heinecke JW; The role of oxidized lipoproteins in atherogenesis;
Free Radic Biol Med 20: 707-727, 1996.
Berry C, Brosnan MJ, Fennell J, Hamilton CA, Dominiczak AF; Oxidative stress
and vascular damage in hypertension; Curr Opin Nephrol Hypertens 10(2): 247-
55,2001.
Blom H, Fowler B, Jakobs C, KocK HG; Disorders of homocysteine metabolism
from rare genetic defects to common risk factors; European Journal of Pediatrics
157, 1998.
64
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Braunwald E; Cardiovascular Medicine at the Turn of the Millennium: Triumphs,
Concerns, and Opportunities; N. Engl. J. Med. 337: 1360-1369, 1997.
Bray MA, Ford-Hutchinson AW, Smith MJH; Leukotriene B4: biosynthesis and
biological activities; In: Piper PJ, ed. SRS-A and Leukotrienes. New York, NY:
John Wiley & Sons, Ltd: 253-270, 1981.
Brown M, Goldstein JL; How LDL receptors influence cholesterol and
atherosclerosis; Sci Am 251: 58-66, 1984.
Burke TW, Kadonaga JT; Drosophila TFIID binds to aconserved downstream
basal promoter element that is present in many TATA-box-defi cient promoters;
Genes Dev 10: 711-724, 1996.
Butler JEF and Kadonaga JT; The RNA polymerase II core promoter: a key
component in the regulation of gene expression; Genes Dev 16: 2583-2592,
2002.
Caterina DR, Zampolli A; From asthma to atherosclerosis — 5-lipoxygenase,
leukotrienes, and inflammation; NEJM 350: 4-7, 2004.
Cooper GR, Myers GL, Smith SJ et al; Blood lipid measurements: variations and
practical utility; JAMA 267: 1652-1660, 1992.
Cordon T, Kannel WB; Premature mortality from comary heart disease: the
Framingham study; JAMA 215:1671-1725, 1971.
Crouse JR, Toole JF, McKinney WM, Dignan MB, Howard Q Kahl FR,
McMahan MR, Harpold GH; Risk factors for extracranial carotid artery
atherosclerosis; Stroke 18(6): 990-996, 1987.
Datta YH, Romano M, Jacobson BC et al; Peptido-leukotrienes are potent
agonists of von Willebrand factor secretion and P-selectin surface expression in
human umbilical vein endothelial cells; Circulation 92: 3304-3311, 1995.
Davydov L, Cheng JW; The association of infection and coronary artery disease:
an update; Expert Opin Investig Drugs 9(11): 2505-2517, 2000.
Descamps OS, Gilbeau JP, Leysen X, Van Leuven F, Heller FR; Impact of
genetic defects on atherosclerosis in patients suspected of familial
hypercholesterolaemia; Eur J Clin Invest 31(11): 958-965, 2001.
65
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Drazen JM, Yandava CN, Dube L et al.; Pharmacogenetic association between
ALOX5 promoter genotype and the response to anti-asthma treatment; Nat Genet
22:168-170, 1999.
Durrington PN; Triglycerides are more important in atherosclerosis than
epidemiology has suggested; Atherosclerosis 141 Suppl 1: S57-62, 1998.
Dwyer JH, Allayee H et al; Arachidonate 5-lipoxygenase promoter
genotype,dietary arachidonic acid, and atherosclerosis; N Engl J Med 350(1): 4-
7, 2004.
Ehara S, Ueda M, Naruko T, Haze K, Itoh A, Otsuka M, et al; Elevated levels of
oxidized low-density lipoprotein show a positive relationship with the severity of
acute coronary syndromes; Circulation 103: 1955-1960, 2001.
Fallest-Strobl CP, Koch DD et al; Homocysteine: A new risk factor for
atherosclerosis; American Family Physician 56, Number 6, 1997.
Fallin D, Cohen A et al; Genetic analysis of case/control Data using estimated
haplotype frequencies: application to APOE locus variation and alzheimer’s
disease; Genome Research 11: 143-151, 2001.
Fallon; Pathology of myocardial infarction and reperfusion; Atherosclerosis and
Coronary Artery Disease: chapter 45,1996.
Ford HAW, Gresser M, Young RN; 5-Lipoxygenase; Annu Rev Biochem 63:
383-417, 1994.
Funk CD, Hoshiko S et al; Characterization of the human 5-lipoxygenase gene;
Proc Natl Acad Sci 86: 2587-2591,1989.
Fuster V; Syndromes of atherosclerosis; American Heart Association, 1996.
Gimbrone AM; Vascular endothelium and atherosclerosis; Vascular injury and
Atherosclerosis (Chapter2): 25-45, 1981.
Glagov S, Hishams B et al; Cerebrovascular disease: a pathologist’s view;
Syndromes of Atherosclerosis, 1996.
Glantz SA, Parmley WW; Passive smoking and heart disease: mechanisms and
risk; JAMA. 273: 1047-1053, 1995.
66
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Goetzl EJ, An S, Smith WL; Specificity of expression and effects of eicosanoid
mediators in normal physiology and human diseases; FASEB J 9: 1051-1058,
1995.
Golden MP, Brock TG; Intracellular compartmentalization of leukotriene
synthesis: unexpected nuclear secrets; FEBS Lett 487: 323-326, 2001.
Goldsmith EM, Humphries RK, et al; “Silent” nucleotide substitution in a P-
thalassemia globin gene activates splice site in coding sequence RNA; Proc Natl
Acad Sci 80: 2318-2322, 1983.
Goodman EJ, Bowman ED, Chanock SJ, et al; Arachidonate lipoxygenase
(ALOX) and cyclooxygenase (COX) polymorphisms and colon cancer risk;
Carcinogenesis 25(12): 2467-2472, 2004.
Gordon D, Qtt J; Assessment and management of single nucleotide
polymorphism genotype errors in genetic association analysis; Pac Symp
Biocomput: 18- 29, 2001.
Gordon T, Castelli WP, et al; High density lipoprotein as a protective factor
against coronary heart disease: The Framingham heart study; Am J Med 62: 707-
714, 1977.
Grech ED, Ramsdale DR, Bray CL, Faragher EB; Family history as an
independent risk factor of coronary artery disease; Eur Heart J 13(10):1311-
1315, 1992.
Hansson GK, Libby P, Schonbeck U, Yan ZQ; Innate and Adaptive Immunity in
the Pathogenesis of Atherosclerosis; Circulation Research 91: 281, 2002.
Haribabu B, Verghese MW, Steeber DA, Sellars DD, Bock CB, Snyderman R;
Targeted disruption of the leukotriene B4 receptor in mice reveals its role in
inflammation and platelet-activating factor-induced anaphylaxis; J Exp Med
192: 433-438, 2000.
Hawkins DJ; Gene structure and expression (Third edition); Cambridge
University Press, 1996.
Hopkins PN, Williams RR; A survey o f 246 suggested coronary risk factors;
Atherosclerosis 40: 1-52, 1981.
Hoshiko S, RMmark O, and Samuelsson B; Characterization of the human 5-
lipoxygenase gene promoter; Proc Natl Acad Sci 87 (23): 9073-9077, 1990.
67
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
In KH, Asano K, et al; Naturally occurring mutations in the human 5-
lipoxygenase gene promoter that modify transcription factor binding and reporter
gene transcription; The American Society for Clinical Investigation 99: 1 ISO-
1137, 1997.
Ingolfsson 10, Sigurdsson G, Sigvaldason H, et al; A marked decline in the
prevalence and incidence of intermittent claudication in Icelandic men 1968-
1986: a strong relationship to smoking and serum cholesterol—the Reykjavik
Study; J . Clin. Epidemiol 47: 1237-1243, 1994.
Janero DR; Therapeutical potential of Vitamin E in the pathogenesis of
spontaneous atherosclerosis; Free Radical Biol Med 11: 129-144, 1991.
Johnson CL, Rifkind BM, Sempos CT, et al; Declining serum total cholesterol
levels among US adults; The National Health and Nutrition Examination
Surveys; JAMA 16; 269(23): 3015-23, 1993.
Kadonaga TJ; The DPE, a core promoter element for transcription by RNA
polymerase II; Experimental and Molecular Medicine 34(4): 259-264, 2002.
Kalin MF, ZumoffB; Sex hormones and coronary disease: a review of the
clinical studies; Steroids 55: 330-52, 1990.
Kario K, Barton Duell P, Matsuo T, et al; High plasma homocyst(e)ine levels in
elderly Japanese patients are associated with increased cardiovascular disease
risk independently from markers of coagulation activation and endothelial cell
damage; Atherosclerosis 157: 441-449, 2001.
Keys A; Seven countries, a multivariate analysis of death and coronary heart
disease; Cambridge: Harvard University Press, 1980.
Kochetov AV, Ischenko IV, Vorobiev DG et al; Eukaryotic mRNAs encoding
abundant and scarce proteins are statistically dissimilar in many structural
features; FEBS Lett 440: 351-355, 1998.
Kruglyak S; The use of a genetic map of biallelic markers in linkage studies; Nat
Genet 17: 21-24, 1997.
Kutach AK, Kadonaga JT; The downstream promoter elementDPE appears to be
as widely used as the TATA box in Drosophila core promoters; Mol Cell Biol
20: 47-54, 2000.
Labarthe DR; Epidemiology and prevention of cardiovascular diseases: a global
challenge. Gaithersburg, Md: Aspen Publishers, Inc.; 1998: 43,127, 1998.
68
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Lagrange I , Kapanidis AN, Tang H et al; New core promoter element in
RNApolymerase II-dependent transcription: Sequence-specific DNA binding by
transcription factor IIB; Genes Dev 12: 34-44, 1998.
Lewontin RC; On measures of genetic disequilibrium; Genetics 120: 849-852,
1988.
Libby P; Changing concepts of atherosclerosis; J Intern Med 247: 349-358,
2000.
Lusis AJ; Genetic factors affecting blood lipoproteins: The candidate gene
approach; J Lipid Res 29: 397-429, 1988.
Lusis AJ; Atherosclerosis; Nature 407: 233 - 241, 2000.
Lusis AJ, Mar R, Pajukanta P; Genetics of atherosclerosis; Annual Reviews of
Genomics and Human Genetics 5: 189-218, 2004.
Maeda K, Okubo K, et al; CDNA cloning and expression of a novel adipose
specific collagen-like factor, apM l; Biochem Biophys Res Commun 221: 286-
Majewski Jacek, Jurg Ott; Amino acid substitutions in the human genome:
evolutionary implications of single nucleotide polymorphisms; Gene 305(2):
167-73, 2003.
Marenberg ME, Risch N et al; Genetic susceptibility to death from coronary
heart disease in a study of twins; N Engl J Med. 330: 1041-1046, 1994.
Matsumoto T, Funk CD, Radmark O, Hoog JO, Jomvall H, Samuelsson B;
Molecular cloning and amino acid sequence of human 5-lipoxygenase; Proc Natl
Acad Sci; 85(10): 3406, 1988.
McGill HC, McMahan CA, Herderick EE et al; Origin of atherosclerosis in
childhood and adolescence; American Journal of Clinical Nutrition 72(5): 1307-
1315, 2000.
McGill HC; Chapter3: Overview; Atherosclerosis and Coronary Artery Disease:
25-41, 1996.
Mehrabian M, Wong J et al; Genetic locus in mice that blocks development of
atherosclerosis despite extreme hyperlipidemia; Circulation Research 89: 125,
2001.
69
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Mehrabian M, Allayee H et al; Identification of 5-lipoxygenase as a major gene
contributing to atherosclerosis susceptibility in mice; Circ Res 91: 120-126,
2002.
Mein AC, Barratt JB et al; Evaluation of single nucleotide polymorphism typing
with invader on PCR amplicons and its automation; Genome Research 10(3):
330-343, 2000.
Meredith IT, Yeung AC, Weidinger FF et al; Role of impaired endothelium-
dependent vasodilation in ischemic manifestations of coronary artery disease;
Circulation 87(Suppl V): 56-66, 1993.
Merten RT; Introducing students to population genetics and the Hardy-Weinberg
Principle; The American Biology Teacher 54(2): 103-107,1992.
Milland J, Christiansen D et al; Translation is enhanced after silent nucleotide
substitutions in A+T-rich sequences of the coding region of CD46 cDNA; Eur J
Biochem, 238(1): 221-230, 1996.
Mohamed AV, Goodricks et al; Subcutaneous adipose tissue release interleukin-
6, but not tumor necrosis factor-a, in vivo; J Clin Endocrinol Metab 82: 4196-
4200, 1997.
Morle F, Lopez B, Henni T and Godet G; alpha-Thalassaemia associated with
the deletion of two nucleotides at position -2 and -3 preceding the AUG codon;
The EMBO Journal 4: 1245-1250, 1985.
Murray CJ, Lopez AD; Alternative projections of mortality and disability by
cause 1990-2020: Global Burden of Disease Study; Lancet 349: 1498-505, 1997.
Napoli C, Armiento D, et al. Fatty streak formation occurs in human fetal aortas
and is greatly enhanced by maternal hypercholesterolemia. Intimal accumulation
of low density lipoprotein and its oxidation precede monocyte recruitment into
early atherosclerotic lesions. J Clin Invest 100(11): 2680-2690, 1997.
National Center for Health Statistics; 5 leading causes of death, United States
2002, all races, both sexes; http://webapp.cdc.gov/cgi-bin/broker.exe, 2004.
Navab M, Fogelman AM, Berliner JA, Territo MC, Demer LL, Frank JS, Watson
Ad, Edwards PA, Lusis AJ; Pathogenesis of atherosclerosis; Am J Cardiol 76,
1995.
Ott J, Rabinowitz D; The effect of marker heterozygosity on the power to detect
linkage disequilibrium; Genetics 147: 927-930, 1997.
70
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Paigen B, Mitchell D, Holmes PA, Albee D; Genetic analysis of strains
C57BL/6J and BALB/cJ for Ath-1, a gene determining atherosclerosis
susceptibility in mice; Biochem Genet 25(11-12): 881-892, 1987.
Patricia CF, David DK, James HS, Patrick EM; Homocysteine: a new risk factor
for atherosclerosis; American Family Physician 6: 1607-1621, 1997.
Pelletier J and Nahum S; Insertion mutagenesis to increase secondary structure
within the 5’ noncoding region of a eukaryotic mRNA reduces translational
efficiency; 1985
Pepine JC, Handberg EM; The vascular biology of hypertension and
atherosclerosis and intervention with calcium antagonists and angiotensin-
converting enzyme inhibitors; Clin Cardiol 24 (Suppl. V), 2001.
Pietrzik K, Bronstrup A; Vitamins B12, B6 and folate as determinants of
homocysteine concentration in the healthy population; European Journal of
Pediatrics 157, 1998.
Pi-Sunyer FX; Medical hazards of obesity; Ann Intern Med 119: 655-660, 1993
Pittilo M; Cigarette smoking, endothelial injury and cardiovascular disease;
International Journal of Experimental Pathology 81: 219-230, 2000.
Poal NR, CooperGJS and Edgar PF; Amylin gene promoter mutations
predispose to Type 2 diabetes in New Zealand M aori; Diabetologia 46(4): 574 -
578, 2003.
Predimank Shah; Pathophysiology of atherothrombosis: role of inflammation;
Inflammatory Disease of Blood Vassels, 2002.
Rai JL, DeMaster EG et al; Cigarette smoke-induced endothelium dysfunction:
role of superoxide anion; J. Hypertens 19: 891-897, 2001.
Radmark Olof; The molecular biologogy and regulation of 5-lipoxygenase; Am J
Respir Crit Care Med 1:161(2): S11-S15, 2000.
Radmark Olof; 5-Lipoxygenase-Derived Leukotrienes: Mediators Also of
Atherosclerotic Inflammation; Arteriosclerosis, Thrombosis, and Vascular
Biology 23: 1140-1150, 2003.
Rannel WB, MCGee D, Gordon T; A general cardiovascular risk profile: the
Framingham study; Am. J. Cardiol 38: 46-57, 1976.
71
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Raul A; Risk factors in coronary atherosclerosis athero-inflammation: the
meeting point; Thrombosis Journal 1: 4-15, 2003.
Renke Maas, Rainer H.Boger; Old and new cardiovascular risk factors: from
unresolved issues to new opportunities; Atherosclerosis Supplements 4: 5-17,
2003.
Rimm EB, Stampfer MJ, Giovannucci E, et al; Body size and fat distribution as
predictors of coronary heart disease among middle-aged and older US men; Am
J Epidemiol 141: 1117-1127, 1995.
Ring WL, Riddick CA, Baker JR, Munafo DA, Bigby TD; Lymphocytes
stimulate expression of 5-lipoxygenase and its activating protein in monocytes in
vitro via granulocyte macrophage colony-stimulating factor and interleukin 3; J
Clin Invest 97: 1293-1301, 1996.
Romano M. and Claria J; Cyclooxygenase-2 and 5-lipoxygenase converging
functions on cell proliferation and tumor angiogenesis: implications for cancer
therapy; FASEB J. 17:1986-1995, 2003.
Ross R, Glomset JA; Atherosclerosis and the arterial smooth muscle cell;
Science 180: 1332-9, 1973.
Ross R; The pathogenesis of atherosclerosis-an update; N Engl J Med 314: 488-
500, 1986.
Ross R; Atherosclerosis—an inflammatory disease; N Engl J Med 340: 115-126,
1999.
Salonen R, Salonen JT; Carotid atherosclerosis in relation to systolic and
diastolic blood pressure: Kuopio Ischaemic Heart Disease Risk Factor Study;
Ann Med 23(1): 23-27, 1991.
Samuelsson B; Leukotrienes: Mediators of immediate hypersensitivity reactions
and inflammation; Science 220: 568-575, 1983.
Scanu AM; Lipoprotein: a genetic risk factor for premature coronary heart
disease; JAMA 267: 3326-3329, 1992.
Segasothy M, and Phillips PA; Vegetarian diet: panacea for modem lifestyle
disease; QJ Med 92: 531-544,1999.
Shatkin AJ; mRNA cap binding proteins: Essential factors for initiating
translation; Cell 40: 223-224,1985.
72
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Sheii LX, Basilion JP, Stanton VP; Single-nucleotide polymorphisms can cause
different structural folds of mRNA; Proc Natl Acad Sci 96: 7871-7876, 1999.
Spanbroek, R et al; Expanding expression of the 5-lipoxygenase pathway within
the arterial wall during human atherogenesis; Proc Natl Acad Sci 100: 1238-
1243, 2003.
Sreachan T, Read PA; Human molecular Genetic (2rd edition); A John Wiley &
Sons INC, 1999.
Stary HC; Evolution of atherosclerotic plaques in the coronary arteries of young
adults; Arteriosclerosis 3: 471 A, 1983.
Stary HC, Chandler B; et al; A Definition of Initial, Fatty Streak, and
Intermediate Lesions of Atherosclerosis; Circulation 89: 2462-2478, 1994.
Steenland K; Passive smoking and risk of heart disease; JAMA 267: 94-99,
1992.
Stemme S, Hansson GK; Immune mechanisms in atherogenesis; Ann Med, Jun.
26(3): 141-146, 1994.
Stenvinkel P; Endothelial dysfunction and inflammation—is there a link? ;
Nephrol Dial Transplant 16: 1968-1971, 2001.
Stephens and Donnelly; PHASE Version 2.02, 2003.
Strachan T, Read PA; Genes in pedigrees; Human Molecular Genetics, chapter 4:
117, 2004.
Stunkard AJ, Wadden TA; Obesity: theory and therapy, Second Edition; New
York: Raven Press, 1993.
Suzuki Y, Tsunoda T, Sese J, et al; Identification and characterization of the
potentialpromoter regions of 1031 kinds o f human genes; Genome Res 11: 677-
684, 2001.
Swain RA, Kaplan-Machlis B; Therapeutic uses of vitamin E in prevention of
atherosclerosis; Altem Med Rev. 4(6): 414-23,1999.
Tailleux A, Duriez P, Fruchart JC, Clavey V ; Apolipoprotein A-II, HDL
metabolism and atherosclerosis; Atherosclerosis 164(1): 1-13, 2002.
permission of the copyright owner. Further reproduction prohibited without permission.
Takais K et al; M echanisms of angiotensin II type 1 receptor blocker for anti-
atherotic effect in monkeys fed a high -cholesterol diet; J Hypertens 21: 361-
369, 2003.
Tell GS, Howard G, McKinney WM; Risk factors for site specific extracranial
carotid artery plaque distribution as measured by B-mode ultrasound; J Clin
Epidemiol.42(6): 551-559, 1989
Terry CF, Loukaci V, Green FR; Cooperative influence of genetic
polymorphisms on interleukin 6 transcriptional regulation; J Biol Chem 275:
18138-18144, 2000.
Van der Wal AC, Becker AE et al; Site of intimal rupture or erosion of
thrombosed coronary atherosclerotic plaque is characterized by an inflammatory
process, irrespective of the dominant plaque morphology; Circulation 89: 36-44,
1994.
Vanecek R, Kagan AR et al; Atherosclerosis of the coronary arteries in five
towns; Bull WHO 53: 509-18, 1976.
Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, et al; The sequence of the
human genome; Science 16; 291(5507): 1304-1351, 2001.
Wasylyk B; Protein coding genes of higher eukaryotes: promoter elements and
trans-acting factors; Maximizing Gene Expression: 79-99, 1987.
Watanabe T, Tokunaga O, Fan J, Shimokama T; Atherosclerosis and
macrophages; Acta Pathol Jpn 39: 473-486, 1989.
Weintraub WS, Klein LW, Seelaus PA, Agarwal JB, Helfant RH; Importance of
total life consumption of cigarettes as a risk factor for coronary artery disease;
Am J Cardiol 55: 669-672, 1985.
Wennmalm A; Nitric oxide (NO) in the cardiovascular system: role in
atherosclerosis and hypercholesterolemia; Blood Press 3(5): 279-82,1994.
Werz Oliver et al; 5-Lipoxygenase: Cellular Biology and Molecular
Pharmacology; Current Drug Targets - Inflammation & Allergy 1: 23-44, 2002.
Zhang YY, Hammarberg T, Radmark O, Samuelsson B, Funk CD, and Loscalzo
J; Analysis of a nucleotide binding site of 5-lipoxygenase by affinity labelling:
binding characteristics and amino acid sequences; Biochem. J. 351: 697-707,
2000.
permission of the copyright owner. Further reproduction prohibited without permission.
Zhao L et al; The 5-lipoxygenase pathway promotes pathogenesis of
hyperlipidemia-dependent aortic aneurysm; Nat. Med. 10: 966-973, 2004
Zhao QW, Minako li, Ken H, Tan CY, Akira T, and Kensuke E; Essential role of
vascular endothelial growth factor in angiotensin II-induced vascular
inflammation and remodeling; Hypertension 44: 264 -270, 2004.
http://www.basic.nwu.edu/biotools/Primer3.html, 2004.
http://www.cbrc.jp/research/db/ TFSEARCH.html, 2004.
http://www.mds.qmw.ac.uk/morbidanatomy/intercal/handoutsetc, 2004
http://www.nucleusinc.com, 2004.
http://cancerweb.ncl.ac.uk/omd
http://snpper.chip.Org/bio/view-snpset/cxme945423/T, 2004.
http://snpper.chip. org, 2004.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Functional analysis of single nucleotide polymorphisms (SNPs) in the 5' regulatory region on the SRD5A2 gene

PDF

Biochemical analysis of somatic mutations in steroid 5alpha-reductase type II in prostate cancer

PDF

Analysis of the HSD3B2 gene in prostate cancer

PDF

Association between single nucleotide polymorphisms in the 3'untranslated region of the SRD5A2 gene and prostate cancer risk

PDF

Glucocorticoids modify signal transducers of bone morphogenetic proteins in osteoblasts: Stimulation of the inhibitory Smad 6 and suppression of the stimulatory Smad 8

PDF

Expression of matrix metalloproteinases and their inhibitors in the muscles of amyotrophic lateral sclerosis and control patients

PDF

A model for the mechanism of agonism and antagonism in steroid receptors

PDF

Generation of mutant tissue inhibitor of metalloproteinases-2 (TIMP-2) in the baculovirus expression system

PDF

Clathrin associated protein (AP) binding motifs in AD5 penton

PDF

Amelogenin domains in a self-assembly process

PDF

Construction and characterization of RRP6 deletion in Saccharomyces cerevisiae

PDF

A review of molecular conjugates and their use in gene therapy with the presentation of a model experiment: Gene therapy with novel fusion proteins that target breast cancer cells

PDF

Biochemical characterization of hydrogen,potassium-ATPase-rich membranes from the gastric parietal cell

PDF

Cyclophilin C is a candidate protein to interact with saposin B using the yeast two-hybrid system

PDF

Establishment and properties of a stable transfected epicardial cell line expressing a dominant negative retinoic acid receptor

PDF

A coactivator complex among GRIP1, CARM1, and TIF1alpha contributes to gene activation directed by androgen receptor

PDF

A TNF alpha-responsive kinase activity may play a key role in IKK activation

PDF

Characterizing the function of murine epididymal secretory protein 1 (ME1) in hematopoietic stem cells

PDF

Molecular studies of the human galactose-1-phosphate uridyltransferase gene

PDF

Identification of the biochemical pathways affected by the anticancer agents Motexafin Gadolinium and Sapphyrin through gene expression profiling

Asset Metadata

Creator Deng, Xuemei (author)

Core Title Analysis of the ALOX5 gene in atherosclerosis

School Graduate School

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Reichardt, Juergen (committee chair), Ou, Jing-Hsiung James (committee member), Tokes, Zoltan A. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-47448

Unique identifier UC11327420

Identifier 1427970.pdf (filename),usctheses-c16-47448 (legacy record id)

Legacy Identifier 1427970.pdf

Dmrecord 47448

Document Type Thesis

Rights Deng, Xuemei

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA