Study On The Polymorphisms Of Asthma Related Genes

Bronchial asthma is a common chronic respiratory disease, characterized by periodic wheezing, cough, and breathlessness. It is caused by complex interactions between genetic and environmental factors. In the past few decades, with the development of industry and the changes of environment, the prevalence and the mortality of asthma have been increasing in most countries. Family and twin studies revealed that there were strong associations of genetic components with asthma. By using positional cloning and genome-wide scans, different chromosomal regions and a number of candidate genes have been found to have linkage with asthma. Among these candidate genes, there were TIM-4,IL-4,IL-13,CD14 genes which are located in the chromosome 5q31-33 region, the IL-4Ra gene which is located in the chromosome 16p11-12 region, and the STAT6 gene which is located in the chromosome 12q14-24 region. Many studies suggest that the imbalance of Thl/Th2 cells and their secretary cytokines may play an important role in the pathogenesis of asthma. And asthma is generally considered a Th2-type disease. TIM-4 gene is one of the members in the TIM gene family, it ligates TIM-1 to provide a costimulatory signal which mediates the proliferation and differentiation of Th2 cells. IL-4 and IL-13 have already been considered the traditional asthma susceptibility genes, and the association studies with asthma have been done in several populations, but the results were inconsistent. IL-4Ra is a component of both the IL-4 and IL-13 receptor complexes, and STAT6 is a key transcription activator involved in IL-4/IL-13 pathway mediated biological function, both of them may be involved in the pathogenesis of asthma. CD14 gene is also located in the asthma susceptibility chromosomal region 5q31.1. It had been reported that a polymorphism-C159T in its promoter was associated with children asthma and total serum IgE concentration. Because asthma is a complex polygenic disease, it is necessary to examine gene-gene interactions for asthma. Although all the six genes have been reported to be linked with asthma in some populations, the effect of gene-gene interactions on asthma among these genes is little known. So study the gene-gene interactions would contribute to a new insight into the pathogenesis of asthma. Generally, just only doing conventional case-control study may lead to higher likelihood of false positive or false negative results as the population stratification. We used both case-control study and family-control study to avoid the false results to clearly indicate the association of these gene polymorphisms with asthma.In this study, we firstly investigated the TIM-4 gene polymorphisms and the association with the susceptibility to asthma by CRS-RFLP, meanwhile, it was also the establishment methodology of CRS-RFLP. Based on the CRS-RFLP method, we then evaluated the single SNP association, haplotype analysis, gene-gene interactions by MDR method of the 8 SNPs in IL-4, IL-13, IL-4Rα, STAT6, CD 14 genes with the development of asthma using case-control study. Furthermore, we used family-based data to do the single SNP association, haplotype analysis, and the gene-gene interactions of the 8 SNPs in the five candidate genes with asthma and total serum IgE levels in a Han population in Middle China. The total study was divided into the following three parts:Part IStudy of the relationship between TIM-4 gene polymorphisms and asthma by CRS-RFLP methodObjective To investigate whether three polymorphism sites of the exon 2 Lys65Lys(G/A), exon 3 Val172Met(G/A), and exon 9 Val365Met(G/A) in T cells immunoglobulin domain and mucin domain protein-4 (TIM-4) are associated with asthma and its phenotype in a Han population from Middle China.Methods The three polymorphisms were detected with created restriction site-restriction fragment length polymorphism (CRS-RFLP) in 185 cases of asthma and 162 healthy controls. The genotype and allele frequencies were calculated and compared between asthma and control groups. Total serum IgE levels were detected by direct chemiluminometric technology. And compared them among different genotype in patients.Results (1) No significant difference in genotype frequencies of GG, GA or allele frequencies of A, G in Lys65Lys(G/A) polymorphism was found between asthma patients and the control subjects (P> 0.05). The AA genotype of Lys65Lys(G/A) was not detected in all the 347 samples. (2) The polymorphism of the Val172Met(G/A) was not detected in our study, all the samples were GG genotype. (3) The polymorphism of the Val365Met(G/A) was not detected in our study, all the samples were GG genotype. (4) No significant difference in total serum IgE levels was found between different genotypes of Lys65Lys(G/A) in patients.Conclusion There were polymorphisms in the exon 2 Lys65Lys(G/A) of TIM-4, but this polymorphism site is not associated with asthma or its phenotype total serum IgE levels in a Han population from Middle China. There was no polymorphism in the exon 3 Val172Met(G/A) or the exon 9 Val365Met(G/A) of TIM-4 in a Han population from Middle China. Part IISingle SNP association and gene-gene interactions of IL-4, IL-13, IL-4Rα, STAT6, and CD14 gene polymorphisms with asthmaObjective To evaluate:1). single SNP association; 2). haplotype analysis in IL-4, IL-13, IL-4Ra genes; 3). and gene-gene interactions of eight single nucleotide polymorphisms (IL-4-C33T, IL-4-C589T, IL-13 R130Q, IL-13 C1923T, IL-4Ra I75V, IL-4Ra Q576R, CD14-C159T, STAT6 C2892T) with the susceptibility to children asthma in a Han population from Middle China.Methods We examined polymorphisms of the eight SNPs (IL-4-C33T, IL-4-C589T, IL-13 R130Q, IL-13 C1923T, IL-4Ra I75V, IL-4Ra Q576R, CD14-C159T, STAT6 C2892T) and performed single SNP association study, haplotype analysis and gene-gene interactions analysis in 479 Chinese children, including 252 asthmatic subjects and 227 healthy controls. Genotyping was performed by created restriction site-restriction fragment length polymorphism (CRS-RFLP) analysis. Haplotype analysis was performed by SHEsis software. Gene-gene interactions were tested using the multifactor dimensionality reduction (MDR) method.Results There were significant differences of IL-13 R130Q and IL-13 C1923T in genotype and allele frequencies distributions in the 8 SNPs between the asthmatic group and control group (P< 0.05). Furthermore, the A allele of IL-13 R130Q and the T allele of IL-13 C1923T were significantly associated with increased risk of asthma (OR= 1.59, 95%CI 1.20-2.09; OR= 1.57,95%CI 1.19-2.08, respectively). None of the other SNPs was associated with asthma. By haplotype analysis, the C-G and T-A haplotypes consisting of IL-13 C1923T and IL-13 R130Q, and the G-A and A-A haplotypes consisting of IL-4Ra I75V and IL-4Ra Q576R were significantly associated with asthma (P< 0.05). There was no significant haplotype in the IL-4 haplotype block consisting of-C33T and-C589T. Using MDR, we detected significant gene-gene interactions with a best six-locus model among IL-4-C33T, IL-13 R130Q, IL-4Ra I75V, IL-4Ra Q576R, STAT6 C2892T and CD14-C159T on the risk of asthma (OR=4.43,95%CI 1.30-15.04; P< 0.001, by 1000 fold permutation test).Conclusions Both single SNP and haplotypes in IL-13 gene polymorphisms were associated with the susceptibility to children asthma in Middle China, indicating that IL-13 gene may play an important role in the development of asthma. The haplotypes in IL-4Ra gene were associated with children asthma in Middle China, suggesting that IL-4Rαgene may influence asthma susceptibility in the form of haplotypes. In addition, the significant gene-gene interactions among IL-4-C33T, IL-13 R130Q, IL-4Ra I75V, IL-4Ra Q576R, STAT6 C2892T and CD14-C159T may increase an individual's susceptibility to asthma in Middle China.PartⅢFamily-based association study of IL-4, IL-13, IL-4Ra, STAT6, and CD14 gene polymorphisms with the susceptibility to asthmaObjective Based on nuclear families data, to investigate 1). single SNP association; 2). haplotype analysis in IL-4, IL-13, IL-4Ra genes; 3). and gene-gene interactions of eight single nucleotide polymorphisms (IL-4-C33T, IL-4-C589T, IL-13 R130Q, IL-13 C1923T, IL-4Ra I75V, IL-4RαQ576R, CD14-C159T, STAT6 C2892T) with the susceptibility to asthma and its phenotype in a Han population from Middle China. Methods Eight single nucleotide polymorphisms (SNPs) containing IL-4-C33T, IL-4-C589T, IL-13 R130Q, IL-13 C1923T, IL-4RαI75V, IL-4RαQ576R, CD14-C159T, STAT6 C2892T were genotyped by created restriction site-restriction fragment length polymorphism (CRS-RFLP) in 126 nuclear families (every nuclear family is composed of one patient and his/her biological parents). Total serum IgE levels were detected by direct chemiluminometric technology. HRR tests were used to investigate the relationship between single SNP and asthma. FBAT software was carried to analyze the associations between haplotypes and asthma, and the associations between single SNP, haplotypes and total serum IgE levels. Gene-gene interactions among the 8 SNPs were tested by the multifactor dimensionality reduction (MDR) software.Results By HRR analysis, none of the 8 SNPs was associated with asthma. By FBAT analysis, the haplotypes in IL-4, IL-13, IL-4Rαgenes were not associated with asthma. The T allele of IL-13 C1923T and the A allele of IL-13 R130Q and were significantly associated with total serum IgE levels (P=0.043; P=0.038). Furthermore, the A allele of R130Q and the T allele of C1923T had higher observed number than expected number in total serum IgE levels, and they could be as risk factors for increased total serum IgE levels. The C-G haplotype consisting of IL-13 C1923T and R130Q, and A-A haplotype consisting of IL-4RαI75V and Q576R were significantly associated with total serum IgE levels (P=0.032; P=0.026). Using MDR, we detected significant gene-gene interactions with a best six-locus model among IL-4-C33T, IL-13 R130Q, IL-4RαI75V, IL-4RαQ576R, STAT6 C2892T and CD14-C159T on the risk of asthma (OR=12.08,95%CI 1.86-78.52; P< 0.001, by 1000 fold permutation test).Conclusions IL-13 gene polymorphisms could influence total serum IgE levels in the forms of single SNP and haplotypes. IL-4Rαgene polymorphisms could influence total serum IgE levels in the form of haplotypes. Gene-gene interactions among IL-4-C33T, IL-13 R130Q, IL-4RαI75V, IL-4RαQ576R, STAT6 C2892T and CD14-C159T may increase an individual's susceptibility to asthma in a Han population in Middle China...