Family- And Haplotype-based Association Study Of TIM-1 And TIM-3 Polymorphisms With Allergic Asthma

Allergic asthma is an immune- related complex disease caused by a combination ofgenetic and environmental factors.The individual genetic factors play an important role inthe development of asthma.By using genome-wide screens or positional cloningtechniques, several chromosomal regions have been found to have linkage with asthma.Among these regions, chromosome 5q31-33 has received the most attention, because itcontains a cluster of cytokines and other immune-related genes and has been repeatedlysuggested to be linked to asthma susceptibility in several populations.A growing body of evidence suggests that the imbalance of T_H1/T_H2 cells and theirsecretary cytokines is relatd with the development of asthma.It has been recently reportedthat the Tims gene family proteins are expressed on T cells and play an important role inregulating T_H1-and T_H2-cell-mediated immunity.In humans, Tim-1 and Tim-3 are the twoimportant members of Tim families.Both of them are located in the chromosome 5q31-33region.Tim-1 is expressed preferentially on T_H2 cells and involved in the development andregulation of T_H2-biased immune responses while Tim-3 is mainly expressed on T_H1 cellsand negatively regulate the T_H1 cell immune responses.In view of the important functionof Tim-1 and Tim-3 in T_H1/T_H2 immune regulation, Tim-1 and Tim-3 have become twogood candidate susceptibility genes for asthma.The association study based the Tim-1 andTim-3 polymorphisms with asthma have been conducted in several populations.However,the results are not unified and repeated because of many reasons.Therefore, therelationships of Tim-1 and Tim-3 polymorphisms and asthma need to be furtherinvestigated.In this study, we firstly investigated the expression of Tim-1 and Tim-3 mRNA inallergic asthma patients and thenby employing the TDT and HRR analysis, we conducted ahaplotype and family-based association study of Tim-1 and Tim-3 ploymorphisms in aChinese population from HuBei province with allergic asthma to examine the hypothesisthat polymorphisms in these two genes are associated with asthma.The total study may bedivided into the following three parts. Partâ… .Expression of Tim-1 and Tim-3 mRNA in PBMC from allergicasthma patientsAIM: To explore the expression of Tim-1 and Tim-3 mRNA in PBMC from allergicasthma patients, for luther clarifying the relationship between Tims genes and asthma.METHOD: A total of 12 patients with mite allergic asthma were collected in random.Asthma was diagnosed according to the criteria formulated by the Respiratory Branchasthma group of Chinese Medical Association in 2003.Another 12 gender and age-matchedhealthy people were selected as the controls.Experiments were divided into 3 groups forasthma, mite stimulation and control.PBMC were separatated by Ficoll separating medium,then taking a 24-hole plate, added 10⁶ PBMC separatated from the patients to the hole ofthe asthma group and the mite stimulation group; Added 10⁶ PBMC from the healthycontrol to the hole of the control group.Additionally, added a final concentration of 20μg/m1 dust mite antigen to the hole of the mite stimulation group.Place the plate in 37℃5% CO₂ incubator to foster for 72 h, then collected the cell and extracted the total RNA.According to the primers sequence from the references, synthesized the Tim-1 and Tim-3primers and then detected the expression of Tim-1 and Tim-3 mRNA by real-timequantitative RT-PCR.RESULT: 1.The relative expression of Tim-1 mRNA in the PBMC of patients with dustmite allergic asthma was 0.27±0.09, significantly higher than the healthy control group,0.13±0.03 (P<0.05); The Tim-1 expression was significantly higher for 0.59±0.22 afterthe PBMC from patients were Stimulated at a final concentration of dust mite antigen for 20μg/m1 for 72 hours.Compared with the control group and asthma group, there aresignificant difference (P<0.01); 2.The Tim-3 mRNA relative expression in the patientswas 0.39±0.09 which is obviously higher than the expression value 0.18±0.06 in thehealthy control (P<0.05).Coincidently, the Tim-3 expression levels also appearedsignificantly increased after stimulation of 20μg/m1 dust mite antigen for 72 hours,reached 0.66±0.22, Which compared with the control group and asthma group, there arealso significant difference (P<0.01);CONCLUSION: The expression Tim-1 and Tim-3 mRNA in the patients with dust miteallergic asthma was significantly increased.Tim-1 and Tim-3 may be involved in theoccurrence and development of human allergic asthma. Partâ…¡.Tim-3 gene polymorphism and the relationship with thesusceptibility to allergic asthmaSectionâ… .Tim-3 sequence determination and identification of polymorphismsAIM: To explore Tim-3 gene promoter and coding region polymorphisms and haplotypecharcteristics in a Chinese Han population from Hubei province and lay a foundation forthe following association study based Tim-3 polymorphisms and some related diseases.METHOD: Randomly collected 30 cases of healthy people and 30 cases of allergicasthma patients from Hubei province as the research subjects.By using the PCRamplification and direct sequenceing technology, detected the polymorphisms in Tim-3promoter, all exons and parts of intron region from the 60 subjects.The sequencing resultswere compared with the HapMap and NCBI database to determinate the Tim-3ploymorphisms characteristic in Hubei Han population.The genotype and allele frequencyfor each locus were compared between the asthma group and control group by Pearsonχ²test or Fisher's exact test.The LD analysis, haplotype construction and selection of tSNPswere conducted by the Haploview software.RESULT: 1.A total of 13 polymorphisms loci were detected in Tim-3 gene region and 5 ofthem were newly discovered.No statistically significant difference were found for thegenotype and allele frequency of each locus between the 30 cases of asthma patients and 30cases heathy people (P>0.05).2.By Comparing with HapMap database, the allelefrequency for rs4704853, rs10515746, rs4704846, rs9313439 between Hubei Hanpopulation and China North Han population and Japanese is not significantly different (P>0.05).However, there is significant difference for the 4 loci allele frequency between theHubei Han population and the Europeans or Africans (P<0.01).3.The LD analysis for 7SNPs with frequency greater than 5% showed that 6 of them has strong linkagedisequilibrium with D' and r² greater than 0.3.The haplotype analysis for the 6 SNPs found5 common haplotypes with frequency greater than 1% in this region.4.rs10053538,rs13170556 and rs9313441 were selected as tSNPs to represent the common variability inthe haplotype block region.CONCLUSION: The distribution of Tim-3 ploymorphisms in Hubei Han population isdifferent from other ethnics.The constructed haplotypes and selected tSNPs can be used forthe next association studies based Tim-3 ploymorphisms with related diseases in Hubei Hanpopulation. Sectionâ…¡.Family-based association study of Tim-3 polymorphisms and allergicasthmaAIM: To investigate the relationship between Tim-3 polymorphisms and haplotypes andallergic asthma and asthma-related phenotype in Hubei Han population.METHOD: 118 complete trios of allergic asthma composed of a patients and theirbiological parents participated were collected from Hubei province, a total of 354 members.By using the PCR-RFLP technology, 3 tSNPs rs10053538, rs13170556 and rs9313441 anda promoter polymorphism rs10515746 were genotyped for the 118 trios; Thehaplotype-tSNPs-based method and family-based association test (TDT, HRR, FBAT) wereemployed to investigate the relationship between Tim-3 polymorphisms and haplotypes andallergic asthma and asthma-related phenotype in Hubei Han population.RESULT: 1.The 4 SNPs of Tim-3 gene single locus TDT and HRR analysis showed thatthere is no linkage disequilibrium between the 4 SNPs and the allergic asthma in Hubei Hanpopulation, P> 0.05.2.The haplotype TDT analysis by using TRANSMIT softwarerevealed that Tim-3 haplotypes are associated with allergic asthma, Globalχ² test,χ² =10.83, P=0.013.Single haplotype TDT results showed that G-G-G haplotype wasundertransmitted from parents to patients (χ²=8.24, P=0.004).The haplotype HRRanalysis showed that G-G-G haplotype had a lower risk (HRR=0.49) and could be used asa protection factor for resistance to allergic asthma.3.FBAT analysis revealed that Tim-3polymorphisms and haplotypes were not associated with asthma phenotype of total serumIgE.CONCLUSION: Tim-3 gene polymorphisms could influence asthma susceptibility in theform of haplotypes, which means Tim-3 gene or the region nearby the Tim-3 may existsome SNPs locus associated with allergic asthma.As a logo the G-G-G Haplotype can beused for asthma susceptibility prediction in Hubei Han population. Partâ…¢.Family-based association study of Tim-1 polymorphisms andallergic asthmaAIM: To investigate the relationship between Tim-1 polymorphisms and haplotypes andallergic asthma and asthma-related phenotype in Hubei Han population.METHOD: 118 complete families of allergic asthma composed of a patients and theirbiological parents participated were collected from Hubei province, a total of 354 members.By using the PCR-RFLP technology, 3 tSNPs (rs4704842,rs953569,rs2134230) and apromoter polymorphism -232 G>A and a exon 4 polymorphism rs45623443 weregenotyped for the 118 trios; The haplotype-tSNPs-based method and family-basedassociation test (TDT,HRR, FBAT) were employed to investigate the relationship betweenTim-1 polymorphisms and haplotypes and allergic asthma and asthma-related phenotype inHubei Han population.RESULT: 1.The 5 SNPs of Tim-1 gene single locus TDT and HRR analysis showed thatthere is no association between the 5 SNPs and the allergic asthma in Hubei Han population,P> 0.05.2.The haplotype TDT and HRR analysis revealed that a haplotype G-A-ins-C-Gin Tim-1 gene region is associated with allergic asthma (P< 0.05).This haplotype wasovertransmitted from parents to asthma patients counld be as a risk factor for allergicasthma with a higher HRR=1.91.3.FBAT analysis revealed that the allele Ins of Tim-1rs45623443 polymorphisms was associated with asthma phenotype of total serum IgE, P<0.05.4.FBAT analysis of Tim-1 haplotypes showed that haplotype G-A-ins-C-G was alsoassociatied with total serum IgE level, P< 0.05.CONCLUSION: Tim-1 gene polymorphisms could influence asthma susceptibility in theform of haplotypes, which means Tim-3 gene or the region nearby the Tim-3 may existsome SNPs locus associated with allergic asthma and high serum total IgE level.As a logo,the G-A-ins-C-G haplotype can be used for asthma susceptibility and serum IgE levelprediction in Hubei Han population.