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Genetic Polymorphism of Prostanoid Receptors, CRTH2 and TBXA2R in Aspirin Hypersensitivity
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | Hae-Sim Park | - |
| dc.contributor.author | Nami Shrestha Palikhe | - |
| dc.date.accessioned | 2019-10-21T07:13:40Z | - |
| dc.date.available | 2019-10-21T07:13:40Z | - |
| dc.date.issued | 2010-02 | - |
| dc.identifier.other | 10607 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/17475 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :의학과,2010. 2 | - |
| dc.description.abstract | -국문요약- 아스피린 천식 과민성 질환에서의 프로스타노이드 수용체인 CRTH2와 TBXA2R의 유전자 다형성 연구 아주대학교 대학원 의학과 나미 슈레스터 팔리케 (지도교수:박 해 심) 연구배경 및 목적: 아스피린 복용은 일부 사람들의 경우 아스피린 과민성 천식, 아스피린 과민성 두드러기, 만성 비염, 아나필락시스 같은 다양한 알레르기 반응을 유발하기도 한다. 본 연구는 아스피린 과민성으로 나타나는 대표적인 두 질환인 과민성 천식과 아스피린 과민성 두드러기에서 프로스타글란딘 D2 수용체, CRTH2,와 프로스타노이드 수용체, TBXA2R 유전자들에 대한 유전체 연구이다. 아스피린 과민성 천식 환자에서 호산구 염증 및 유입이 아스피린 내성 천식 환자에 비해 두드러지게 나타남을 고려해 볼때, 프로스타글란딘 D2 수용체인 CRTH2 유전자의 유전자 다형성에 따른 기능적 변화를 통한 호산구의 활성화가 아스피린 과민성 천식의 병인기전에 중요한 역할을 담당할 것으로 가정하고 CRTH2 유전자의 유전자 다형성 조사 및 아스피린 과민성 천식과의 연관성 연구를 수행하였다. 더욱이, 천식 및 아토피와의 연관성이 보고된 TBXA2R 유전자의 경우, 그 유전자의 유전자 다형성에 따른 기능 변화가 아스피린 과민성 두드러기의 병인기전과 관련되는지를 유전자 연관성 연구로 조사하였다. 재료 및 방법: 본 연구는 아주대 병원에 내원한 107명의 아스피린 과민성 천식 환자, 115명의 아스피린 내성 천식 환자, 그리고 133명의 정상대조군을 대상으로 CRTH2 유전자의 유전자 다형성 조사 및 연관성 연구를 수행하였다. TBXA2R 유전자의 경우, 아주대 병원에 내원한 167명의 아스피린 과민성 급성 두드러기 환자, 316명의 아스피린 과민성 만성 두드러기 환자, 그리고 265명의 정상대조군을 대상으로 수행하였다. 두 유전자의 유전형은 SNAPshot ddNTP primer extention 키트를 사용하여 결정되었다. 두 유전자의 유전자 다형성에 따른 기능 변환 연구는 luciferase reporter assay와 electrophoretic mobility shift assay로 수행하였다. 결과: 아스피린 과민성 천식 환자군에서 아스피린 내성 천식 환자군에 비해 높은 eotaxin-2 생성이 관찰되었다 (p=0.034). 아스피린 과민성 천식 환자군과 아스피린 내성 천식 환자군에서 CRTH2 -466T>C 유전자 다형성의 대립유전자 빈도의 차이가 통계적으로 유의하게 관찰되었다 (p<0.05). 또한, 아스피린 과민성 천식 환자군에서, CRTH2 -466TT 유전자형을 가진 환자들의 경우 -466CT 또는 -466CC 유전자형을 가진 환자들 보다 혈청 eotaxin-2 생성이 높게 관찰되었다. 인간 폐 상피세포를 이용한 시험관내 기능 연구를 통해 -466T 대립유전자는 낮은 luciferase activity를 보였고(p<0.001), 낮은 mRNA 발현을 보이고, 반면 높은 eotaxin-2 생성이 관찰되었다(p=0.003). EMSA 실험을 통해, -466T 대립유전자에 -466C 대립유전자보다 더 높은 친화도를 가지고 결합하는 전사인자를 확인하였다. TBXA2R -4684T>C 유전자 다형성의 대립유전자 빈도의 차이는 아스피린 과민성 급성 두드러기 환자군과 정상 대조군에서 통계적으로 유의하게 관찰되었다 (p= 0.015). 아스피린 과민성 급성 두드러기 환자군에서, TBXA2R -4684T>C 유전자 다형성은 아토피와 연관성(p= 0.013)을 보였지만, 아스피린 과민성 만성 두드러기 환자군에서는 아토피와 연관성을 보이지 않았다. 인간 비반 세포주인 HMC-1을 이용한 시험관내 기능 연구를 통해 TBXA2R -4684T 대립유전자는 낮은 luciferase activity를 보였고, EMSA 실험을 통해 -4684T 대립유전자에 -4684C 대립유전자보다 더 높은 친화도를 가지고 결합하는 전사인자를 확인하였다. 또한, 아스피린 과민성 급성 두드러기 환자들은 정상 대조군에 비해 낮은 thromboxane B2 생성을 보였다 (p=0.016). 결론: 본 연구를 통해, 프로스타글란딘 D2 수용체, CRTH2,와 프로스타노이드 수용체, TBXA2R 유전자들의 유전자 다형성들이 아스피린 과민성과 연관성이 있으며, 특히 CRTH2 유전자는 아스피린 과민성 천식과 TBXA2R 유전자는 아스피린 과민성 급성 두드러기와 연관성이 있음을 확인하였다. 핵심어: 아스피린 과민성, 천식, 급성 두드러기, 유전자 다형성, 호산구 | - |
| dc.description.tableofcontents | TABLE OF CONTENTS ACKNOWLEDGEMENT I ABSTRACT II TABLE OF CONTENTS VI LIST OF FIGURES IX LIST OF TABLES XI ABBREVIATIONS XII PART-I 1 GENETIC POLYMORPHISM OF CRTH2 IN ASPIRIN INTOLERANT ASTHMA 1 I.INTRODUCTION 2 II. MATERIALS AND METHODS 5 A. Study subjects and phenotyping 5 B. SNP identification and genotyping 6 C. Statistical analysis 7 D. In vivo functional study of CRTH2 -466T>C by measurement of eotaxin-1 and eotaxin-2 using enzyme-linked immunosorbent assay 7 E. Preparation of the CRTH2 construct and dual luciferase assay 8 F. Nuclear extract preparation and electrophoretic mobility shift assay 10 G. Preparation of CRTH2-coding sequence (CDS) (-466T or -466C) and in vitro functional study of eotaxin-2 production in A549 cells and mRNA expression 11 III. RESULTS 13 A. Clinical characteristics of the CRTH2 study subjects 13 B. Genotype and allele frequencies of the CRTH2 gene with the AIA phenotype 15 C. Eotaxin level according to the CRTH2 gene polymorphism 18 D. Eotaxin-2 level according to severity of AIA patients based on classification of FEV1 (%) 18 E. Endogenous expression of CRTH2 from various cell lines 20 F. Dual luciferase activity of CRTH2 -466T>C polymorphism 21 G. Effects of CRTH2 -466T>C gene polymorphisms on transcriptional activity 22 H. CRTH2 expression and in vitro production of eotaxin-2 from CRTH2 transfected A549 cells 23 IV. DISCUSSION 31 PART-II 35 GENETIC POLYMORPHISM OF TBXA2R IN ASPIRIN INTOLERANT ACUTE URTICARIA 35 I. INTRODUCTION 36 II. MATERIALS AND METHODS 39 A. Study subjects and phenotyping 39 B. SNP identification and genotyping 40 C. Statistical analysis 40 D. Preparation of the TBXA2R construct and dual luciferase assay 41 E. Electrophoretic mobility shift assay 42 F. In vivo measurement of TXB2 production from sera of AIAU and NC 43 III. RESULTS 45 A. Clinical characteristics of the TBXA2R study subjects in AIAU 45 B. Genotype and allele frequencies of the TBXA2R gene with the AIAU phenotype 45 C. Dual luciferase activity of TBXA2R -4684T>C polymorphism 50 D. TBXA2R -4684T>C gene polymorphisms on transcriptional activity 50 E. TXB2 production according to TBXA2R -4684T>C gene polymorphism 56 IV. DISCUSSION 57 V. CONCLUSION 60 REFERENCES 61 국문요약 70 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | Genetic Polymorphism of Prostanoid Receptors, CRTH2 and TBXA2R in Aspirin Hypersensitivity | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.department | 일반대학원 의학과 | - |
| dc.date.awarded | 2010. 2 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 568541 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000010607 | - |
| dc.subject.keyword | genetic Polymorphism | - |
| dc.subject.keyword | prostanoid Receptors | - |
| dc.subject.keyword | CRTH2 | - |
| dc.subject.keyword | TBXA2R | - |
| dc.subject.keyword | hypersensitivity | - |
| dc.description.alternativeAbstract | - ABSTRACT - Genetic Polymorphism of Prostanoid Receptors, CRTH2 and TBXA2R in Aspirin Hypersensitivity Background and Objective: Aspirin ingestion can induce a wide range of clinically recognized allergic reactions, including aspirin intolerant asthma (AIA), also called aspirin exacerbated respiratory disease (AERD), aspirin-intolerant urticaria/angioedema (AIU), chronic rhinitis, and anaphylaxis. We studied two major aspirin hypersensitivity, AIA and AIAU in an association with prostanoids receptor CRTH2 and TBXA2R. The human CRTH2 gene (official name GPR44) encodes a G protein-coupled chemoattractant receptor molecule which is expressed on Th2 cells including other allergy related cells like eosinophils, basophils and monocytes. Its importance in inflammatory cell activation and recruitment in vitro and in vivo has been continuously reported in several studies. Considering the fact that eosinophilic infiltration is more pronounced in AIA patients than it is in ATA patients, we hypothesized that activation of eosinophils via dysregulation of the CRTH2 gene may play an important role in AIA and may be an important marker of this entity. Therefore, this case control study was designed to determine if variation in the CRTH2 gene confers elevated risk for developing AIA. Human Thromboxane A2 (TBXA2), is another eicosanoid product induced by cyclooxygenase may be as important mediator in aspirin hypersensitivity as it can induce bronchoconstriction and bronchohyperresponsiveness. TBXA2 exerts its action by interacting with the G protein-coupled thromboxane A2 receptor (TBXA2R). Based on previous association of TBXA2R with asthma and atopy, we investigated whether genetic polymorphisms of the TBXA2R gene are associated with AIAU phenotype through a case-control of three study subjects. Materials and Methods: The three studies groups 107 patients with AIA, 115 patients with ATA, and 133 normal healthy controls (NC) were recruited from Ajou University Hospital, Suwon, Korea. We genotyped two polymorphisms of CRTH2 located in front of exon-2 (upstream of the ATG) using a primer extension method and the SNAPshot ddNTP primer extension kit. (Applied Biosystems, Foster City, CA, USA). In case of TBXA2R gene polymorphism, three subject groups (167 patients with AIAU, 316 patients with AICU, and 265 patients with normal controls) were enrolled at Ajou University Hospital in Suwon, Korea. Two polymorphisms of TBXA2R (-4684T>C and 795 T>C) were genotyped using a primer extension method and the SNAPshot ddNTP primer extension kit (Applied Biosystems, Foster City, CA, USA). The functional effect of polymorphism of CRTH2 and TBXA2R were analyzed by luciferase reporter assay, electrophoretic mobility shift assay and ELISA. Results: AIA patients had significantly higher serum eotaxin-2 levels than did those with ATA (p=0.034). A significant difference in the genotype frequencies of CRTH2 -466T>C was detected between AIA and ATA patients (p<0.05). The serum eotaxin-2 level was significantly higher in AIA patients carrying the TT genotype than those with the CT and CC type (p<0.05). In vitro functional study demonstrated that the -466T allele had lower luciferase activity (p<0.001) and lower mRNA expression with higher production of eotaxin-2 (p=0.003) in human lung epithelial cells. EMSA showed that CRTH2 -466T produced a specific band with a higher affinity than CRTH2 -466C had. AIA patients exhibited a significantly higher prevalence of paranasal sinusitis and nasal polyps compared to ATA patients (p<0.001 and p<0.001, respectively). The serum eotaxin-2 level was significantly higher in AIA patients than in ATA patients (p=0.034) in our study, while no difference was noted in the eotaxin-1 level. Furthermore, the genotype frequency of TBXA2R -4684T>C was significantly different AIAU and NC on both co-dominant and recessive analysis models. Specifically, AIAU patients showed a significantly higher frequency of the homozygous TT genotype of TBXA2R -4684T>C compared to NC (p=0.015). In addition, there was significant association of atopy status according to TBXA2R -4684T>C polymorphism. AIAU patients carrying TT genotype showed significantly higher atopy status compared to CT or CC type (p=0.013) but not in AICU patients (P>0.05). In vitro functional study demonstrated that the -4684T allele had lower luciferase activity (p<0.001) in HMC-1 cells. EMSA showed that TBXA2R -4684T produced a specific band with a higher affinity than TBXA2R -4684C. There was a significant difference between AIAU vs. NC with respect to serum Thromboxane B2 production (TXB2). AIAU patients showed a significantly lower TXB2 production compared to patients with NC (p=0.016). Conclusion: These study suggest that genetic variability of prostanoid receptors CRTH2 and TBXA2R may be associated with aspirin hypersensitivity, in particular CRTH2 ？466T allele showed higher frequency and increased serum and cellular eotaxin-2 production through lowering of CRTH2 expression in AIA and TBXA2R -4684T allele showed higher frequency and lowered TXB2 production through lowering of TBXA2R expression in AIAU. __________________________________________________________________________ Key words: aspirin hypersensitivity, respiratory disease, acute urticaria, genetic polymorphism, eosinophils, eotaxin, thromboxane. | - |
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