Evaluation of the impact of polymorphisms on candidate genes of allergic rhinitis and asthma on disease outcomes in the singapore population

EVALUATION OF THE IMPACT OF POLYMORPHISMS
ON CANDIDATE GENES OF ALLERGIC RHINITIS AND
ASTHMA ON DISEASE OUTCOMES IN THE SINGAPORE
POPULATION








LIANG XIAOHUI

NATIONAL UNIVERSITY OF SINGAPORE

2006




EVALUATION OF THE IMPACT OF POLYMORPHISMS
ON CANDIDATE GENES OF ALLERGIC RHINITIS AND
ASTHMA ON DISEASE OUTCOMES IN THE SINGAPORE
POPULATION







LIANG XIAOHUI

(BACHELOR OF MEDICINE)






A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF OTOLARYNGOLOGY

NATIONAL UNIVERSITY OF SINGAPORE

2006

i

ACKNOWLEDGEMENTS

First and foremost, I would like to express my appreciation to my supervisor, Associate
Prof. Wang De Yun, for providing me the opportunity to carry out this interesting project.
It was he who brought me to this area of research. He provided guidance, encouragement
and support throughout my postgraduate study. Without his help, it would have been
impossible for me to complete this project on time.
I would like to express my sincere gratitude to Dr. Wai Cheung from the Department of
Pediatrics at Oregon Health & Sciences University in the US for his contribution to this
project as well as his valuable suggestions. I would like to deeply appreciate Dr. Heng
Chew Kiat

from the Pediatrics

Department at National University of Singapore, Prof.
Bing Lim and Dr. Liu Jianjun from the Genome Institute of Singapore, for providing
useful comments, materials and technical assistance. I would also like to thank Tan Sin
Lam from the Institute for Infocomm Research for spending much time in helping me
analyze the data in chapter 8.
I would like to thank my good friends Li Hongzhe, Bai Jing, Foong Kok Heng, Huang
Zhili and He Xuelian. Whenever I meet with any difficulties, they have always given
their generous help. The same goes to Dr. Lau Quek Choon and Priya Kadam for
spending their precious time polishing the English.

I owe a special gratitude to my parents, husband and daughter for their constant love,
support and encouragement during my academic pursuits.
I would also like to thank the National University of Singapore for the postgraduate
research scholarship.

ii

Table of Contents

Acknowledgments i
Table of Contents ii
Summary vii
List of Tables ix
List of Figures xiv
List of abbreviations xvii
Publication from this study xx

CHAPTER 1. INTRODUCTION 1
1.1 Definition of atopy and atopic diseases 1
1.1.1 Atopy 1
1.1.2 Allergic rhinitis 2
1.1.3 Allergic asthma 3
1.1.4 Atopic dermatitis 4
1.1.5 Relationship between atopy and atopic diseases 4
1.2 Pathophysiology and immunology of atopy 6
1.2.1 Basic concepts in immunology 6
1.2.2 Hypersensitivity 7
1.2.3 Pathophysiology of atopic diseases 7
1.2.4 Candidate genes implicated in the pathophysiology of atopy 11
1.3 Genetics of atopy 12

1.3.1 Basic concept in genetics used in the study 13
1.3.2 Approaches to study the genetic components of atopy 15
1.3.3 General strategies for defining susceptibility genes 18
1.4 Procedures in genetic research: susceptibility genes 20
1.5 Summary of candidate gene on atopy 22
1.5.1 Susceptibility regions 23
1.5.2 Susceptibility genes 28
1.6 Environment factors influencing atopic diseases 39
1.6.1 Hygiene hypothesis 39
1.6.2 Plausible mechanism of the hygiene hypothesis 40
1.7 Atopic diseases in Singapore 41
1.8 Importance of the study 45
1.9 Hypothesis of this study 46
1.10 Objective of this study 46
1.11 Value of this study 47

iii
CHAPTER 2. MATERIALS AND METHODS 48
2.1 Study population 48
2.2 DNA extraction 48
2.2.1 Blood collection 49
2.2.2 DNA extraction 49
2.3 PCR (Polymerase Chain Reaction) 51
2.3.1 Primer design 51
2.3.2 PCR reaction 52
2.4 DNA agarose gel electrophoresis 52
2.5 RFLP Restriction Fragment Length Polymorphism 53
2.6 DNA sequence analysis 53
2.7 Cell culture 55
2.7.1 Cell culture materials 55

2.7.2 Environment for cell culture 55
2.7.3 Culture of adherent cell lines 55
2.7.4 Culture of suspension cell lines 56
2.7.5 Refreshing cells 56
2.7.6 Freezing cells 57
2.7.7 Cell counts 57
2.8 Cloning 58
2.8.1 Primer design 58
2.8.2 PCR amplification and purification 59
2.8.3 Vectors 60
2.8.4 Digestion 61
2.8.5 DNA purification 62
2.8.6 Ligation 62
2.8.7 Preparation of competent cells 63
2.8.8 Transformation 64
2.8.9 Minipreparation of plasmid DNA 64
2.8.10 Plasmid selection 65
2.8.11 Large-scale preparation of endotoxin free plasmid DNA 66
2.9 Transfection 68
2.9.1 Transfection by the Lipofectamine Reagent 68
2.9.2 Transfection by electroporation 69
2.10 Luciferase activity assay 70
2.11 Immunofluorecent staining of HepG2 Cells 71
2.12 Flow cytometry 72
2.13 sCD14 ELISA 72
2.14 Total Serum IgE 73
2.15 Statistical analysis 73

iv

2.15.1 Determination of sample size for a case-control design 73
2.15.2 Hardy-Weinberg equilibrium 75
2.15.3 Statistical methods 76

CHAPTER 3. ASSOCIATION BETWEEN CD14 POLYMORPHISMS AND
ATOPY 78
3.1 Introduction 78
3.1.1 Role of CD14 78
3.1.2 Current studies on CD14 polymorphisms 79
3.1.3 Objectives 81
3.2 Results 81
3.2.1 CD14 sequences 81
3.2.2 tIgE measurement 83
3.2.3 sCD14 measurement 85
3.2.4 CD14/-159 polymorphism 87
3.2.5 CD14/-550 polymorphism 93
3.2.6 CD14/-1145 polymorphism 95
3.2.7 CD14/-1359 polymorphism 99
3.2.8 CD14/-1619 polymorphism 103
3.2.9 Linkage disequilibrium among polymorphisms on the CD14 promoter 105
3.2.10 Association between haplotypes and atopic phenotypes 105
3.2.11 Association between sCD14 and total serum IgE levels 106
3.2.12 Functional study 107
3.2.13 Sequence analysis of the CD14 exon 123
3.3 Discussion 124

CHAPTER 4. ASSOCIATION BETWEEN IL-4 POLYMORPHISM AND ATOPY
132
4.1 Introduction 132
4.1.1 Role of IL-4 132

4.1.2 Current studies on IL-4 polymorphisms 133
4.1.3 Objectives 136
4.2 Results 135
4.2.1. IL-4 Sequence 136
4.2.2 RFLP results of the IL-4 polymorphism 137
4.2.3 Sequence analysis of the IL-4 polymorphism 138
4.2.4 IL-4/-590 polymorphism 139
4.2.5 Association between IL-4/-590 polymorphism and tIgE levels 140
4.2.6 Association between IL-4/-590 polymorphisms and atopic phenotypes 141
4.3 Discussion 141


v

CHAPTER 5. ASSOCIATION BETWEEN β
2
-ADRENERGIC RECEPTOR
POLYMOR-PHISMS AND ATOPY 146
5.1 Introduction 146
5.1.1 Role of β
2
-adrenergic receptor 146
5.1.2 Current studies on B2AR polymorphisms 148
5.1.3 Objectives 151
5.2 Results 152
5.2.1 B2AR sequences 152
5.2.2 RFLP results of the B2AR polymorphisms 152
5.2.3 B2AR polymorphisms 154
5.2.4 B2AR polymorphisms in three populations 156
5.2.5 Association between B2AR polymorphisms and tIgE levels 157

5.2.6 Association between B2AR polymorphisms and atopic phenotypes 157
5.2.7 Linkage disequilibrium between B2AR polymorphisms 158
5.2.8 Association between haplotypes of B2AR polymorphisms and atopy
phenotypes 159
5.3 Discussion 160

CHAPTER 6. ASSOCIATION BETWEEN TLR4 POLYMORPHISMS AND
ATOPY 163
6.1 Introduction 163
6.1.1 Role of TLR4 163
6.1.2 Current studies on TLR4 polymorphisms 165
6.1.3 Objectives 168
6.2 Results 168
6.2.1 TLR4 sequences 168
6.2.2 PCR results 169
6.2.3 Sequence analysis of the TLR4 polymorphisms 169
6.3 Discussion 170

CHAPTER 7. ASSOCIATION BETWEEN IL-18 POLYMORPHISMS AND
ATOPY 172
7.1 Introduction 172
7.1.1 Role of IL-18 172
7.1.2 Current studies on IL-18 polymorphisms 175
7.1.3 Objectives 176
7.2 Results 177
7.2.1 IL-18 sequences 177
7.2.2 Sequence analysis of the IL-18 polymorphisms 178
7.2.3 IL-18 polymorphisms 180
7.2.4 Association between IL-18 polymorphisms and tIgE levels 182

vi

7.2.5 Association between IL-18 polymorphisms and atopic phenotypes 182
7.2.6 Association between haplotype and atopy phenotypes 183
7.2.7 Functional study 184
7.3 Discussion 194

CHAPTER 8. SEARCHING FOR CANDIDATE GENES ASSOCIATED WITH
ATOPY 200
8.1 Background 200
8.2 Results 203
8.2.1 Association between haplotypes and tIgE levels 203
8.2.2 Association between haplotypes and atopic diseases 206
8.3 Discussion 210

CHAPTER 9. CONCLUSIONS 212

REFERENCES 217

vii

SUMMARY
Atopy is a disorder with strong familial tendency, starting usually in childhood or
adolescence when patients become sensitized and produce IgE antibodies in response to
ordinary allergens. However, the complex mechanisms of inheritance, from genetic
predisposition of atopy to atopic (allergic) diseases, are still incompletely understood.
Many candidate genes have been identified using positional cloning and/or candidate
gene techniques. Recent data suggest that the pathogenesis of atopic diseases is complex
and might be caused by gene-gene and/or gene-environmental interactions.

This study aimed to investigate the association between candidate gene polymorphisms
and atopic phenotypes in the Singapore population of Chinese (n=331), Malay (n=51)
and Asian Indian (n=101) ethnic backgrounds. Further, transcriptional activity of some
polymorphisms was investigated using a reporter gene assay system, in order to
determine whether these polymorphisms affect candidate gene function.

In this study, we were able to detect the polymorphisms on the CD14 promoter at
positions -159, -550, -1145, and -1359; the IL-4 promoter at -590; the IL-18 5’ non-
trancription region at positions -137, +113 and +127; and the β
2
-adrenergic receptor
(B2AR) at the amino acid positions 16 and 27 (Arg16Gly and Gln27Glu) in all three
ethnic groups. There was strong linkage disequilibrium in the CD14 and IL-18
polymorphisms. In the Chinese, a novel polymorphism (CD14/-550) was identified,
however, the two commonly reported polymorphisms on Toll-like receptor 4 (TLR-4) at
the amino acid positions 299 and 399 (Asp299Gly and Thr399Ile) were not observed.

viii
The results showed that there exist obvious ethnic differences in the allele frequencies of
the various polymorphisms on candidate genes, such as: TLR-4, IL-4 and B2AR. No
association was observed between all polymorphisms evaluated and atopy-related
phenotypes, e.g. total IgE (tIgE) levels and allergic rhinitis and asthma, except
association between CD14 polymorphisms and allergic asthma in Malays.

The influence of polymorphisms on CD14 promoter and IL-18 5’ non-trancription region
on transcriptional activity were investigated by reporter assay in different cell-lines
(THP-1, U937, HepG2 and HeLa). The results showed that polymorphisms on the CD14
promoter did not show any obvious effects on its transcriptional activity. On the contrary,
polymorphisms on the IL-18 5’ non-trancription region were found to influence
transcription activity.

This study reports no association between some reported polymorphisms of candidate
genes in patients with atopy and allergic diseases in Singapore population. One could
argue that it is due to the possible difference in the study population, this clearly indicate
that predisposition to atopy is influenced by complex immune processes, including
interactions of multiple genes, interactions of environmental and genetic factors and
population heterogeneity. Future studies would be needed to identify the key genes for
atopic phenotypes and to investigate the interactions between genetic and environmental
factors that influence the complex trait of allergic diseases.



ix

LIST OF TABLES
1. Summary of association studies on candidate genes for atopy 37
2. Characteristics of the study subjects 48

3. Primers to assay the polymorphisms on candidate genes 51
4. Primers for PCR to amplify inserted DNA 59
5. Estimation of sample size by allele frequency and anticipated odds ratio (AOR)74
6. Summary of association studies on CD14 polymorphisms 81
7. Concentration for standard curve and raw data for sCD14 86
8. Genotypes distribution of CD14/-159 in non-atopic and atopic subjects and Hardy-
Weinberg equilibrium 89
9. Comparison of CD14/-159 allele frequency among three different ethnic groups90
10. Serum sCD14 levels by CD14/-159 genotypes in Chinese group 91
11. Geometric mean (95% CI of the mean) serum tIgE(kU/l) by CD14/-159
genotypes in three different ethnic groups 91
12. Association between CD14/-159 polymorphism and atopic phenotypes 92
13. Genotypes distribution of CD14/-550 in non-atopic and atopic subjects and Hardy-
Weinberg equilibrium 94

14. Serum soluble CD14 levels by CD14/-550 genotypes in Chinese group 94
15. Geometric mean (95% CI of the mean) serum tIgE (kU/l) by CD14/-550
genotypes 95
16. Association between CD14/-550 genotype frequency and atopic phenotypes 95

x

17. Genotypes distribution of CD14/-1145 in non-atopic and atopic subjects and Hardy-
Weinberg Equilibrium 97
18. Comparison of CD14/-1145 allele frequency among three different ethnic groups-97
19. sCD14 levels by CD14/-1145 genotypes in Chinese group 98
20. Geometric mean (95% CI of the mean) serum tIgE(kU/l) by CD14/-1145
genotypes in three ethnic groups 98
21. Association between CD14/-1145 polymorphism and atopic phenotypes 99
22. Genotypes distribution of CD14/-1359 in non-atopic and atopic subjects and Hardy-

Weinberg equilibrium 101
23. Comparison of CD14/-1359 allele frequency among three different ethnic groups101
24. sCD14 levels by CD14/-1359 genotypes in Chinese group 102
25. Geometric mean (95% CI of the mean) serum tIgE(kU/l) by CD14/-1359
genotypes in three different ethnic groups 102
26. Association between CD14/-1359 polymorphism and atopic phenotypes 103
27. Pair-wise linkage disequilibrium of CD14 gene 105
28. Association between CD14 haplotypes and sCD14, tIgE and atopic diseases 106
29. Match results for the CD14 promoter 107
30. Increase of RLA for CD14 promoter with different polymorphisms in
HepG2 120

31. Increase of RLA for CD14 promoter with different polymorphisms in
HepG2 (LPS stimulation) 120


xi

32. Increase of RLA for CD14 promoter with different polymorphisms in
THP-1 121

33. Increase of RLA for CD14 promoter with different polymorphisms in
U937 121
34. Summary of association studies on IL-4/-590 polymorphism 135
35. Frequency of IL-4/-590 genotype in non-atopic and atopic group and
comparison of allele frequency among three ethnic groups 139

36. Geometric mean serum total IgE level (IU/ml) (95%CI of the mean) by
IL-4/-590 genotypes in three ethnic groups 140

37. Multiple comparisons between IL-4/-590 polymorphisms and serum tIgE
in Chinese atopic group 140
38. Association between IL-4/-590 polymorphism and atopic phenotypes in three
ethnic groups 141
39. Summary of association studies on B2AR polymorphisms and atopy 150
40. Genotypes distribution of B2AR polymorphism at amino acid 16 in non-atopic and
atopic subjects and Hardy-Weinberg equilibrium 155

41. Genotypes distribution of B2AR polymorphism at amino acid 27 in non-atopic and
atopic subjects and Hardy-Weinberg equilibrium 156
42. Comparison of B2AR allele frequency among three ethnic groups 156
43. Geometric mean serum total IgE level (IU/ml) (95%CI) by polymorphisms
at position 16 and 27 of the B2AR in three ethnic groups 157

44. Association between B2AR polymorphism at position 16 and 27 and
atopic diseases in three ethnic groups 158

xii

45. Linkage disequilibrium of B2AR 158
46. Association between B2AR haplotypes and atopic phenotypes 159
47. Summary of association studies on TLR-4 polymorphisms 167

48. Summary of association studies on IL-18 polymorphisms 176

49. Genotypes distribution of IL-18 polymorphism at amino acid 27 in non-atopic and
atopic subjects and Hardy-Weinberg equilibrium 181

50. Pair-wise linkage disequilibrium of IL-18 181
51. Comparison of IL-18 allele frequency among three different ethnic groups 181

52. Mean of serum tIgE levels (IU/ml) (95% CI of the mean) by IL-18 genotypes
between atopy and non-atopy groups in three races 182
53. Association between IL-18 polymorphisms and atopic phenotypes 183
54. Association between IL-18 haplotypes and atopic phenotypes 183
55. Transcription factor binding sites of IL-18 promotor 184
56. Increase of RLA for IL-18 promotor with different polymorphisms
in HepG2 189

57. Increase of RLA for IL-18 promotor with different polymorphisms in
Hela 190

58. Increase of RLA for IL-18 promotor with different polymorphisms in
U937 191

59. Increase of RLA for IL-18 promotor with different polymorphisms in
THP-1 191

xiii
60. Association between haplotypes and tIgE levels 204
61. Association between haplotypes and atopic phenotypes 206

xiv

LIST OF FIGURES

1. Summary of pathophysiology on atopic diseases 11
2. Flowchart of genetic research in atopy and atopic diseases 22
3. Plausible mechanism of hygiene hypothesis 41
4. Procedure for treating blood 49
5. Basic methods in genotypes detection 54
6. Hemocytometer 58
7. pGL3 vector map 60
8. pRL-CMV vector map 61
9. Basic steps of subcloning 68
10. sIgE results from clinical laboratory 84
11. tIgE results using UniCAP 85
12. sCD14 standard curve 87
13. CD14/-159 RFLP results 88
14. CD14/-159 sequencing results 89
15. CD14/-550 sequenceing results 93
16. CD14/-1145 polymorphism results 96
17. CD14/-1359 polymorphism results 100
18. Allele specific PCR results of CD14/-1619 104

xv

19. No correlation between sCD14 and serum tIgE level 107
20. CD14 immunofluorecent staining of HepG2 Cells 109

21. Results of CD14-FITC binging to THP-1 cells 110
22. Results of CD14-FITC binging to U937 cells 111
23. Results of CD14-FITC binging to U937 cells stimulated by LPS for 36 hours-112

24. Structure of 8 constructed plasmids 113
25. A constructed plasmid transformed into E.Coil on the LB plate 114
26. Gel electrophoresis to confirm the inserted DNA (A) 115
27. Gel electrophoresis to confirm the inserted DNA (B) 115
28. Gel electrophoresis to confirm the inserted DNA (C) 116
29. Sequence results of 6 constructed plasmids 117

30. Sequence results for 2 constructed plasmids 119
31. Relative luciferase activity (RLA) of transfected cells 122
32. CD14 exon PCR results 123
33. Sequencing results of two novel polymorphisms on CD14 exon 124
34. Biological effect of IL-4 133
35. IL-4 PCR results 137
36. IL4/-590 RFLP results 122
37. IL-4/-590 sequencing results 138

xvi

38. Effects of β
2
-adrenergic receptor 147
39. Primary amino acid sequence and proposed membrane topography of
the human B2AR 148
40. B2AR PCR results 153
41. B2AR RFLP results 154
42. Signaling pathway of Toll-like Receptors 164

43. TLR4 in pathway of immune response 165
44. TLR4 PCR results 169
45. TLR4 sequencing results 169
46. The role of IL-18 173
47. IL-18 PCR results 178
48. IL-18/-137 sequencing results 179
49. IL-18/ +113 and IL-18/+127 sequencing results 180
50. PCR results of IL-18 inserted DNA 186
51. Gel electrophoresis to confirm the IL-18 inserted DNA 186
52. Sequencing results of 2 constructed plasmids 188
53. Luciferase reporter assay for human IL-18 promoter region. 193





xvii

LIST OF ABBREVIATIONS
5’-UTR 5’ untranslated regions
Aa amino acid
Amp ampicillin
APCs antigen presenting cells

AR airway responsiveness

B2AR β
2
-adrenergic receptor

BHR bronchial hyperreponsiveness

bp base pairs
o
C degree celcius
CC competent cell. e.g. DH-5α
CD cluster of differentiation
CHRM1 cholinergic receptor muscarinic 1

DNA deoxyribonucleic acid

DMSO dimethyl sulfoxide
dNTP deoxyribonucleoside triphosphates
EMSA electrophoretic mobility shift assays
FBS fetal Bovine Serum
g gram or gravitational force
GM-CSF granulocyte-macrophage colony-stimulating factor
HeLa cervical epithelial cell line
HepG2 hepatocellular cell line
HLA human lymphocyte antigen

xviii
ICAM intercellular adhesion molecule
IFN interferons
IL interleukin
IL17F interleukin-17 family
IRAK interleukin-1 receptor– associated kinase
Kb kilo bases
LB Luria-Bertani broth
LBP LPS binding protein

LFA lymphocyte function-associated antigen

LPS lipopolysaccharides
MAP3K mitogen-activated protein 3 kinase
mg milligram
MHC major histocompatibility complex
min minute
ml milliliter
mM millimolar
NF-κB nuclear factor-kappa B
NEAA non-essential amino acid
PBS Phosphate-buffered saline
PCR polymerase chain reaction
PMA phorbol 12-myristate 13-acetate
RFLP Restriction Fragment Length Polymorphism
SDS sodium dodecyl sulfate

xix

sCD14 soluble CD14
sIgE specific IgE
SNPs single nucleotide polymorphisms
STATS signal transducers and activators of transcription
TAP transporter of antigen peptides
TCR T-cell receptor
tIgE total IgE
Th1 T-helper type 1
Th2 T-helper type 2
THP-1 human monocytic Mono Mac cell line

TLR-4 Toll-like receptor 4
TNF Tumor necrosis factor
TRAF-6 tumor necrosis factor receptor–associated factor 6
U937 histiocytic lymphoma cell line
µg microgram
µl microlitre
UV ultraviolet
VEGF Vascular endothelial growth factor










xx

Publications from this study

Abstracts for conferences

(1) Xiao Hui Liang, Wai Cheung, Chew Kiat Heng, De Yun Wang. Association between
CD14 promoter polymorphism and atopy in Singapore population. BioMedical Asia
2001, September 2001. Singapore.

(2) Xiao Hui Liang, Wai Cheung, Chew Kiat Heng, De Yun Wang. Polymorphisms in
the Beta-2 adrenoceptor gene and atopic diseases. Third combined annual scientific

meeting Life sciences for Singapore. November 2001. Singapore.

(3) Xiao Hui Liang, Wai Cheung, Chew Kiat Heng, De Yun Wang. Single nucleotide
polymorphisms of CD14 gene: results from atopic patients in Singapore Chinese.
Third combined annual scientific meeting Life sciences for Singapore. November
2001. Singapore.

(4) Xiao Hui Liang, Wai Cheung, Chew Kiat Heng, De Yun Wang. Association of atopy
and IL-4 promoter gene in Singapore population. 6
TH
NUS-NUH Annual Scientific
Meeting. August 2002. Singapore.

(5) Xiao Hui Liang, Wai Cheung, Chew Kiat Heng, De Yun Wang. Association of CD14
promoter polymorphisms with atopy phenotypes in Singapore population. American
Academy of Allergy Asthma and Immunology 60
TH
anniversary meeting. March
2003. Denver, USA.

(6) Xiao Hui Liang, Wai Cheung, Chew Kiat Heng, De Yun Wang. Association of
Polymorphisms of Candidate Genes on Chromosome 5q31-33 and Atopic Diseases in
Singapore. World Allergy Organization Congress-XVIII ICACI. September 2003.
Vancourer, Canada.

(7) Xiao Hui Liang, Wai Cheung, Chew Kiat Heng, De Yun Wang. Association of CD14
promoter polymorphisms with atopy phenotypes in Singapore population. 7
TH
NUS-
NUH annual scientific meeting. October 2003. Singapore.

(8) Xiao Hui Liang, Wai Cheung, Chew Kiat Heng, De Yun Wang. Association of
polymorphisms of candidate genes on chromosome 5q31-33 and atopic diseases in
Singapore. 7
TH
NUS-NUH annual scientific meeting. October 2003. Singapore.

(9) Xiao Hui Liang, Wai Cheung, Chew Kiat Heng, Chun Wei Li, and De Yun Wang.
Ethnic variation of IL-4/-590 polymorphism among Chinese, Malays and Asian
Indians in Singapore. XIX World Allergy Organization Congress. June 26-July 1,
2005. Munich, Germany


xxi

(10) Xiao Hui Liang, Bing Lim, Chew Kiat Heng, Wai Cheung, Jian Jun Liu, Chun
Wei Li and De Yun Wang. CD14 promoter polymorphisms are not associated with
atopic phenotypes. XIX World Allergy Organization Congress. June 26-July 1, 2005.
Munich, Germany.


Publications in peer view journals

(1) Xiao Hui Liang, Wai Cheung, Chew Kiat Heng, De Yun Wang. Absent of the
toll-like receptor 4 gene polymorphisms Asp200Gly and Thr399Ile in
Singaporean Chinese. Therapeutics and clinical risk management, 2005: 1(3):
243-246.

(2) Xiao Hui Liang, Wai Cheung, Chew Kiat Heng, De Yun Wang.Reduced
transcriptional activity in individuals with interleukin-18 gene variants detected

from functional but not association study. Biochemical and Biophysical Research
Communications,2005: 338: 736-741.

(3) Xiao Hui Liang, Wai Cheung, Chew Kiat Heng, Jian Jun Liu, Chun Wei Li, Bing
Lim, De Yun Wang. CD14 promoter polymorphisms have no functional
significance and are not associated with atopic phenotypes. Pharmacogenetics and
Genomics, 2006: 16: 229-236.

(4) Ethnic variation of IL-4/-590 polymorphism among Chinese, Malays and Asian
Indians in Singapore. DNA Sequencing ( to be submitted)

(5) Polymorphisms in the β
2
-adrenergic receptor gene and atopic diseases in
Singapore population (to be submitted)










1

CHAPTER 1
INTRODUCTION

Atopic diseases represent a global health problem due to their increasing high prevalence,
especially in industrialized countries. Although environmental factors play important
roles, there is a strong genetic predisposition in the development of atopy and atopic
diseases (Mackay and Rosen, 2001; Arshad, 2002; Marshall, 2004).

1.1 Definition of atopy and atopic diseases
1.1.1 Atopy
Allergy is an inappropriate and harmful response to a normally harmless substance and is
usually caused by proteins, called allergens, including pollens, dust mites, animal dander,
food and so on. Atopy is a kind of allergy which refers to an inherited predisposition to
produce IgE antibodies (Arshad, 2002). The term “atopy” comes from the Greek word
Atopos which means “out of place”. Atopy is thus defined as a personal or familial
tendency to produce IgE antibodies in response to low doses of allergens, usually proteins,
and to develop typical symptoms such as asthma, rhinoconjunctivitis or eczema
(Johansson et al., 2001; Roitt and Delves, 2001). Although these allergens target different
organs, in most patients they are characterized by elevated total IgE levels. At least 25-
30% of the population is atopic (Arshad, 2002). But the existence of atopy may or may
not lead to development of atopic diseases including asthma, allergic rhinitis, and atopic
dermatitis. Genes and environment factors both play an important role in development of
these diseases.

2


According to the definition from “Revised Nomenclature of Allergy for Global Use”
made by the World Allergy Organization in October 2003, the term, atopy, should be
revised to describe the genetic predisposition to become Ig-E sensitized to allergens
commonly occurring in the environment, and cannot be used until an IgE sensitization
has been documented by IgE-antibodies in serum or by a positive skin prick test
Johansson et al., 2004). In our study, we defined the atopy according to the specific

serum IgE (sIgE) to five common antigens, namely Bermuda grass (G2), Aspergillus
fumigatus (M3), Blatella germanica (Cockroach) (I16), Dermatophagoides pteronyssinus
(house dust mite) (D1) and Dermatophagoides farinea (house dust mite) (D2). Subjects
were classified into atopic and non-atopc groups on the basis of serum specific IgE (sIgE)
levels. Subjects with sIgE levels equal or more than 0.35 ku/l to at least one of the
inhalant allergens belonged to the atopic group, and the others belonged to the non-atopic
group.

In one word, atopy was defined as a positive skin prick test (SPT) or a positive serum
specific IgE (equal or more than 0.35U/ml) to at least one of the common inhalant
allergens tested.

1.1.2 Allergic rhinitis
Allergic rhinitis is associated with an excessive generation of specific IgE and
characterized by one or more of the following symptoms: anterior nasal symptoms of
pruritus, sneeze, discharge and stuffiness, and an associated loss of sense of smell and