Case Control And Family Based Study On Relationship Between TIM4 Gene Polymorphisms And Atopic Asthma

Asthma is a chronic inflammatory disease, characterized by periodic obstruction of theairways and respiratory symptoms which are wheeze, cough and breathlessness. Theprevalence of it has been increasing in most countries. Interaction between geneticsusceptibility and environmental exposure is essential to the phenotypic expression ofasthma. Several linkage studies have suggested that a region on chromosome 5q31-33contains genetic variations associated with asthma. Disturbance in the T helper type 1 (Th1)/T helper type 2 (Th2) balance is thought to play a critical role in the pathophysiology ofasthma. The gene family of T-cell immunoglobulin domain and mucin domain (TIM)proteins, which is located in 5q33, has been shown to play a role in the T cell-mediatedimmune responses. It contains three members in human: TIM1, TIM3 and TIM4. TIM1 isexpressed on all activated T cells and, upon CD4+ T-cell polarization, at a higher level onTh2 cells than on Th1 cells. TIM3 is preferentially expressed on terminal differentiated Th1cells, which down regulates the T cells after conjunction with its ligand. TIM4, which is thenatural ligand of TIM1, is expressed mainly on dendritic cells (DCs) and macrophages.DCs express TIM4 that ligates TIM1 to drive the proliferation and expansion of Th2 cells.The recent reports showed that high expression of TIM4 induced naive CD4+ T cells tobecome Th2 cells, and caused the occurrence of hypersensitivity. These data support animportant role for TIM4 in the promotion of Th2-cell responses, thus TIM4 is supposed tobe a candidate research gene of asthma. Association Studies have showed that several SNPsof TIM1 and TIM3 were associated with asthma susceptibility in some, but not in allpopulations. Natalie S Page found that the major haplotype of TIM4 gene was associated with childhood atopic dermatitis in Caucasian population. So far, the associations betweenTIM4 promoter and asthma have not been studied as there are no published papers lookingat these associations.In the present study, based on the determined TIM4 gene polymorphisms in asthmapatients of Chinese Han population, we investigated the association of TIM4 genepolymorphisms with asthma, and explored the potential biology functions of TIM4 genevariations. Our purpose was to study the relationship between TIM4 gene polymorphismsand asthma. The total study is divided to the following three parts:PartⅠDetermination the sequence and SNPs of TIM4 promoterand exon region in asthma patients of Chinese Han populationObjective To investigate the distribution characters of T cell immunoglobulin domainand mucin domain protein 4(TIM4) promoter region, exons, 3' untranslated regionpolymorphisms in asthma patients of Chinese Han population, and the haplotypefrequences of TIM4 promoter。Methods The promoter region, exons, 3' untranslated region of TIM4 was resequencedby PCR-sequencing method, and linkage disequilibrium was analyzed by JLIN software.Results Six single nucleotide polymorphisms(SNPs) were found, which were located inpromoter region, exon 2 and exon 9. Three SNPs (-1609G＞A, -153 T＞C, 43746 C＞T)were discovered for the first time, the minor allele frequencies were 0.10, 0.15, 0.05respectively. The A allele frequency of SNP rs6874202 was higher in asthma patients thanin common people. The frequencies distribution of rs6874202 and rs6882076 were differentamong different races (P＜0.05 ) . In addition, linkage disequilibrium among the SNPs ofthe promoter region of TIM4 was found, and GGTG was the predominant haplotype.Conclusion The SN-P distribution characters of TIM4 in Chinese Han population were different from other races. The SNP rs6874202 might be genetically related to asthma.There are four SNPs in the promoter region of TIM4 in the asthma patients of Chinese Hanpopulation, which are in linkage disequilibrium.PartⅡAssociation study of TIM4 gene polymorphisms with asthmaand its phenotypeObjective To investigate the association between the SNPs / haplotypes of TIM4 withasthma and its phenotype.Methods The polymorphisms (rs6874202 & rs6882076) were detected by polymerasechain reaction-restriction fragment length polymorphism (PCR-RFLP) in 208 asthmapatients and 232 healthy controls. The allele / genotype frequencies of rs6874202 &rs6882076 in asthma and control groups were compared. Total serum IgE levels weremeasured using direct chemiluminometric technology. The associations between rs6874202& rs6882076 and total serum IgE levels were analyzed. Ten SNTs (TIM4 new1,rs6874202, TIM4new2, rs6882076, 8579G＞A, rs7724832, rs7700944, rs1345617,rs1345618 and TIM4 new3) were detected by polymerase chain reaction-restrictionfragment length polymorphism (PCR-RFLP) in 118 nuclear families. TDT test were carriedto analyze the associations between the detected SNPs with asthma and total serum IgElevels. The haplotypes and haplotype frequencies were constructed and calculated using theHaploview software. The association between TIM4 haplotype and asthma were analyzedby Transmit software.Results The distribution of genotype and allele frequencies of rs6874202 between theasthma patients and healthy controls showed significant difference (P＜0.01 ). A allele waspreferencially transmitted from parents to their off springs, which also indicated that A allele was associated with asthma. Other SNPs were not associated with asthma. All thedetected SNPs were not associated with total serum IgE levels. There were two haplotypeBlocks in TIM4 gene. GGTGGG was the prevalent haplotpye (Frequency: 0.647) inBlock 1 and GACAGG haplotype was in positive correlation with asthma, GGTAGGhaplotype was in negtive correlation with asthma. The common haplotypes of Block2 werenot associated with asthma.Conclusion rs6874202 was associated with asthma, the risk of A allele carrier was1.91 times of the G allele carrier. GACAGG haplotype is in positive correlation withasthma, and may increase asthma susceptibility.PartⅢStudy on the transcription activity of TIM4 promoter byluciferase reporter gene systemObjective To construct two luciferase reporter vectors containing two different TIM4gene promoter, and study the effect of different TIM4 gene promoter sequence on thetranscription of luciferase in 16HBE cell line, evaluate the regulation of SNP rs6874202 onthe promoter transcription activity of TIM4 gene.Methods GACA and GGCA haplotype PCR products were generated by overlap PCRmethod. The PCR products were directly cloned into luciferase reporter pGL3-Basic. TakepRL-TK as control plasmid, pGL3-GACA, pGL3-GGCA and pGL3-Basic were instantlytransfected into 16HBE cell lines, the relative activity of luciferase were detected after 48hours.Results Two luciferase reporter vectors containing different TIM4 gene promoter weresuccessfully constructed confirmed by restriction enzymes digestion and nucleotidesequencing, and they were different from each other only in single nucleotide. Two vectors both can be expressed in 16HBE cells, and the relative activities of luciferase weresignificantly stronger than pGL3-Basic. The reporter vectors pGL3-GACA, pGL3-GGCAhad different relative luciferase activities, the relative luciferase activity of pGL3-GACAwas 1.45 times as much as pGL3-GGCA.Conclusion Two luciferase reporter vectors pGL3-GACA, pGL3-GGCA containingdifferent TIM4 gene promoter were successfully constructed. Compared with G allele, theA allele of SNP rs6874202 could inhance the transcription activity of TIM4 promoter,which may increase the susceptibility to asthma.