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 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. i 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 ii 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 iii 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 iv CHAPTER 5. ASSOCIATION BETWEEN β2-ADRENERGIC RECEPTOR POLYMOR-PHISMS AND ATOPY----------------------------------------------------146 5.1 Introduction --------------------------------------------------------------------------146 5.1.1 Role of β2-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 v 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 vi 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’ nontrancription region at positions -137, +113 and +127; and the β2-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 (TLR-4) at the amino acid positions 299 and 399 (Asp299Gly and Thr399Ile) were not observed. vii 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. 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Am J Respir Crit Care Med. 2000; 161: 1655-9. 251 [...]... 2001) On the other hand, the release of the inflammatory cytokines leads to the “late phase reaction”, which primarily involves the recruitment and activation of eosinophils, Th2 cells, macrophages and neutrophils (Rothenberg, 1998) Once the late phase is initiated, eosinophages become one of the major mediators of chronic inflammation in allergic asthma (Maddox and Schwartz, 2002) In addition, interleukin... expression and secretion of pro-inflammatory mediators and cytokines (Bousquet et al., 2003) An initiative in collaboration with WHO termed Allergic Rhinitis and its Impact on Asthma (ARIA) has been developed to assess the relationship between asthma and 5 rhinitis (Bousquet et al., 2001) In this project, we studied disease candidate genes for allergic rhinitis and asthma together 1.2 Pathophysiology and. .. al., 1999) Allergic rhinitis and allergic asthma are common respiratory diseases that often coexist and the allergic mechanisms in the two diseases have similar patterns Both of them are associated with raised levels of IgE (Sherrill et al., 1999) and the concept of “one airway, one disease has arisen However not all rhinitis patients develop asthma and not all asthma patients with rhinitis, there are... or eosinophils in different tissues or organs in human induce different clinical manifestations of allergy, including: asthma, allergic rhinitis and atopic eczema The summary of the whole pathophysiology of atopic diseases is shown in Figure 1 Figure 1: Summary of pathophysiology on atopic diseases The four main steps include antigen presentation, Th1 and Th2 differentiation, IgE production and granules... (3) environmental factors - environment is necessary for expression of the disease phenotype (FitzGerald et al., 2001) In the following section, basic concepts in genetics and methods for studying genetic contribution to atopic diseases are presented and association studies on candidate genes in atopic diseases are summarized 1.3.1 Basic concepts in genetics used in the study 1.3.1.1 Glossary of genetic... obstruction, rhinorrhea, sneezing and itchy nose) on most days during the past year (Bousquet et al., 2001) Allergic rhinitis was defined if atopy coexisted 1.1.3 Allergic asthma The condition termed asthma is difficult to define satisfactorily The difficulty arises from poor understanding of its causes, pathophysiology, and also a lack of a specific marker of the disease Asthma is currently defined as... the triggers include indoor allergens such as mold, house dust mite, cockroach and animal dander The symptoms are the same as those of seasonal allergic rhinitis but are experienced throughout the year (Roecken et al., 2004) Allergic rhinitis can occur at any age and affects approximately 20% of the young population (Bradley and Mccluskey, 1997) In one word, rhinitis was defined as the occurrence of. .. helper type 2 (Th2) based on interactions with the microenvironment In the presence of interleukin 12 (IL-12), interleukin 18 (IL-18), or interferons γ (IFN-γ), it 8 differentiates into Th1 cells This evolution is mediated by a mechanism that depends on signal transducer and activator of transcription-1 (STAT-1) and the T-bet transcription factor Whereas, in the presence of interleukin 4 (IL-4) (which comes... example, atopy often precedes asthma and increases the risk of developing asthma 10-20 times (Holgate, 1999) There are also close associations among atopic diseases For example, 75% of asthmatics have rhinitis and 20% to 40% of rhinitis patient suffer from asthma (Palma-Carlos et al., 2001) In another epidemiological study, rhinitis was reported to occur in up to 80% of patients with asthma (Leynaert... De Yun Wang Association of polymorphisms of candidate genes on chromosome 5q31-33 and atopic diseases in Singapore 7TH 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 . 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. EVALUATION OF THE IMPACT OF POLYMORPHISMS ON CANDIDATE GENES OF ALLERGIC RHINITIS AND ASTHMA ON DISEASE OUTCOMES IN THE SINGAPORE POPULATION LIANG. 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