Kawasaki Disease Immunity Related Gene Expression Difference Analysis In Peripheral Blood Mononuclear Cells After LPS Stimulated

medium-sizedarteries,especially coronary arteries,which is characterized by the activation of the immune system and the extensive damage of the vascular endothelial system.Although the cause of the disease is not clear,studies have shown that the pathogenesis is about that the abnormal activation in immune system and abnormal function of immunomodulatory in KD.In the first part of this paper,we focus on the abnormal mutation of immuno-related genes in KD children by target area capture and next generation sequencing technology.The second part,using bacterial lipopolysaccharide to stimulate peripheral blood mononuclear cells,analyzed the difference of the expression of susceptible genes related to KD immunoregulation.Kawasaki disease(KD)is an acute systemic vasculitis occurring inPart1:Identification of susceptibility genes related to immunity associated with Kawasaki disease by targeted enrichment of genomic region sequencing techniqueObjective To discover susceptibility genes related to immunity associated with Kawasaki disease,providing experimental evidences of the pathogenesis of KD in the future.Methods According to the inclusion criteria,199 KD patients and 99 outpatients for health examination were recruited in Shanghai children’s hospital from November 2015 to March2017.472 single nucleotide polymorphisms associated with KD susceptibility genes and 512 genes in T cell receptor signaling pathway,toll-like receptor signaling pathway,Cytokine receptor interaction,TGF-beta signaling pathway were selected as targeted genes,and customed exons capture sequencing chip.Then use Illumina Hi Seq X10 for high-throughput sequencing.The sequencing datas were used for bioinformatics analysis and burden test in order to find out susceptibility genes associated with KD.Results After burden test,there was a significant difference between the KD group and the normal group that seven genes SNV groups collection of seven SNPs(p <0.05).Among them,the relationship between CD84,IL-10 and SMAD2 genes and the pathogenesis of KD have been reported.The first discovery of PIK3 CG,PRKCQ and PTPN6 genes was significantly associated with the pathogenesis of KD.Conclusions Genes CD84,ELF5,IL-10,PIK3 CG,PRKCQ,PTPN6,SMAD2 associated with the pathogenesis of KD.Part2:Kawasaki disease Immunity related gene expression difference analysis in peripheral blood mononuclear cells after LPS stimulatedObjective Kawasaki disease(KD)peripheral blood leukocytes(PBMCs)were stimulated in vitro to focus on the KD immune-related susceptibility genes,observing whether the susceptibility gene mutations affect the gene expression,which provided a basis for further research on the pathogenesis of KD.Methods According to the inclusion criteria,20 children with KD and 20 healthy children with physical examination in Shanghai Children’s Hospital were selected as study subjects which were divided into KD group and control group.Peripheral blood mononuclear cells(PBMCs)of two groups were extracted.After bacterial lipopolysaccharides(LPS)stimulated cells and culture medium were isolated.The intracellular RNA was extracted and the difference of transcriptome was analyzed by RNA sequencing.Then an expression quantitative trait locus(e QTL)analysis in20 KD samples was conducted to examine thedifferentially expressed genes.To confirm the findings in e QTL analysis,the culture medium levels of 13 cytokines were detected before and after LPS stimulation in all 10 samples.Results1)After RNA sequencing,six abnormal expression genes were found to be associated with KD,of which mi RNA500 and PLXND1 were significantly associated with immune system development and functional regulation(P <0.01),GIT1 and RUNX3 were significantly associated with cardiovascular system development and function(P <0.01).2)Correlation analysis of e QTL-associated DNA revealed that the mutation which affected genes’ expression in KD including PIK3CD(rs11121484)C / T(P = 0.04),PIK3CD(rs1141402)G / A and ITPR3(rs2229634)(P = 0.01),PRKCZ rs34108055 G / C(P = 0.02),MAP2K1 rs1438386 G / A(P =0.01),ITGB2 rs2280965 C / T C / T(P = 0.04).3)After the analysis of the KD immuno susceptible gene in the first part,it was found that the abnormal expression of IL-10 and PTPN6 genes were significantly different between the KD group and the normal group.4)After stimulation with LPS,the important immunosuppressant cytokines were significantly decreased,SCD25(Z =-2.79,P = 0.02)and IL-10(Z =-3.10,P <0.01)compared with the control group in KD group.Conclusions The immunoregulatory related genes and cell molecules have significant differences in the expression of mimicking inflammatory reactions which are associated with the pathogenesis of KD.