The Study Of SNP And Methylation Of Immune Related Molecules And Cell Imbalance Mechanism In Uygur Patients With Pigeon Breeder’s Lung

Objective: The aim of study is to discuss SNPs and methylation of immune related molecules and cell imbalance Mechanism in Uygur Patients with Pigeon Breeder’s lung(To investigate the Correlation between single nucleotide polymorphisms in the gene of MIP-1α、HLA-DRB5 gene methylation、CD4+CD25+ Foxp3+Treg cell and susceptibility in Uygur Patients with Pigeon Breeder’s lung) Methods: 1) Based on inclusion and exclusion criteria, 92 subjects including 32 patients with PBL, 30 negative controls with history of exposure to pigeons and 30 normal controls without pigeons contact. DNA of all blood samples were extracted, according to the sequence information about SNP loci appendix in UCSC database, MassARRAY technology was used to design the primers, then completed the sample preparation、 PCR amplification、 SAP and single base extension, SNPs in the gene of MIP-1α was analyzed using mass spectrometry; 2) DNA of all blood samples were extracted, bisulfite conversion, PCR amplification, transcription in vitro and RNase a specific enzyme cut, parallel matrix assisted laser desorption ionization time of flight mass spectrometry technique was used to detect HLA-DRB5 gene methylation status; 3) Based on inclusion criteria, 32 patients with PBL, including 15 acute-phase, 17 chronic-phase, 30 negative controls with history of exposure to pigeons; all blood samples were collect, Foxp3+ CD4+Treg cell and lymphocyte subsets were detect with flow cytometry; 4) SPSS 17.0 was used for statistical analysis to baseline information, SAS statistical software was used for the others, Trend chi-square test was used for being not in Hardy-Weinberg equilibrium. Results: 1) Compared with normal control group, PBL group and negative control group had significantly higher concentration of MIP-1α; 2) 24 loci in MIP-1α gene were surveyed, 7 of them were found to be polymorphic(Figure); 3) Hardy-Weinberg equilibrium test: the genotypes frequency distribution of MIP-1α SNPs rs1049191, rs1049195, rs3210166, rs1130374 and rs5029407 were not in Hardy-Weinberg equilibrium in PBL group and normal group(P < 0.05), however, the genotypes frequency distribution of MIP-1α SNPs rs1063340 was in Hardy-Weinberg equilibrium in PBL group and normal group;(P>0.05); 4) Genotype distribution of MIP-1α SNPs rs1049191, rs1049195, rs3210166, rs1130374, rs190805628 and rs5029407 were significantly different among the three groups(P<0.05); However, the distribution frequencies of the 2 alleles of all those were no significantly different among the three groups(P>0.05); 5) combining normal control group and negative control group to one group, Compared with PBL group, Genotype distribution of MIP-1α SNPs rs1049191, rs1049195, rs3210166, rs1130374 and rs5029407 were significantly different among the two groups(P<0.05); 6) Linkage Disequilibrium Measures: ten groups strong LD existed among seven loci in MIP-1α gene, Haplotype C-C-G-G-C-C-C and Haplotype T-A-G-A-C-G-T might have an association with the susceptibility of PBL in Chinese Uygur population; 7) Eighteen sites(CpG1 、 CpG2/3/4 、 CpG5/6/7 、 CpG8/9/10 、CpG11/12/13、CpG14、CpG16/17/18/19) of HLA-DRB5 fragment were detected, there were no significant differences in methylation rate in all sites of HLA-DRB5 fragment among the three groups(P > 0.05); 8) Compared with normal control group, the CD4+CD25+ FOXP3+Treg cell and CD4+CD25+ cell were significantly lower than in the control group(P<0.01); In the PBL groups, particularly the acute-phase group(P<0.01); There were no significant differences in CD4+CD25+ cells between the PBL groups; 9) Compared with normal control group CD4+ T cell percentages and CD4+/CD8+ ratios were significantly lower than in the control group(P<0.01) and CD8+ T lymphocyte percentage was significantly higher than in the control group; 10) In peripheral blood from the PBL groups, the CD4+/CD8+ ratio was positively correlated with the CD4+ CD25+FOXP3+Treg cell(r=0.864, P<0.05) and CD4+/CD25+cell(r=0.34, P < 0.05) percentages. Conclusion: 1) MIP-1α rs1049191, rs1049195, rs3210166, rs1130374, rs1063340, rs190805628 and rs5029407 were found to be polymorphic in Uygur; 2) Contacting the pigeon may lead to higher concentration of MIP- 1 alpha, however, higher concentration of MIP- 1 alpha might have not an association with the susceptibility of PBL; 3) MIP-1α SNPs might have an association with the susceptibility of PBL in Chinese Uygur population. Those SNPs might have influence to the expression or activity of MIP-1α. 4) CC genotypes 1n MIP-1α SNPs rs1049191, rs1049195, r S3210166, rs5029407 may increase the risk of suffering from PBL, GG genotypes 1n MIP-1α SNPs rS1130374 may increase the risk of suffering from PBL It provide the basis for disease prevention; 5) Which there were no differences in methylation rate of HLA-DRB5 gene among three group means the relationship between HLA-DRB5 methylation and PBL was not found; 6) It suggested that decrease of CD4+ CD25+ FOXP3+Treg cell might play an important role in the occurrence of PBL. the imbalance of Th1/Th2 is the main molecular biology mechanism in Uygur patients with PBL.The percentages of Tregs and the changes in T cell subsets in peripheral blood were analyzed via flow cytometry. It will help to assist in the diagnosis of PBL. At present, there is no related reports at home and abroad.