Association Study Of Ischemic Stroke Associated With Toll-like Receptor Signaling Pathway As Well As The Polymorphisms Of Its Key Genes In Chinese Population

Objectives:The aim of this.study was to screen the biological pathways(Toll-like receptor pathway)as well as its key genes(TRAF6,TLR4)related to the susceptibility of ischemic stroke with different Traditional Chinese medicine(TCM)syndrome,and to estimate the association between the TRAF6,TLR4 genes polymorphisms and TCM syndrome as well as lipid levels in Chinses Han ischemic stroke patients.We hope it can provide molecular biology evidence for standardized and objective of TCM diagnosis.Methods:In this study,Gene Set Enrichment Analysis(GSEA)was used to screen out the functional key genes and biological pathways of ischemic stroke,and the key genes associated with the proliferation and apoptosis of ischemic stroke were chosen.Then,a case-control study was performed.184 ischemic stroke cases were recruited from the neurology inpatient department of GuangDong Province Hospital Of Traditional Chinese Medicine and 114 healthy controls were recruited at the same period of time and the same place.A questionnaire survey(Case Report Form,CRF)was conducted on all the subjects in this study.The Sanger genotyping method was carried out to analysis the 5 single nucleotide polymorphisms(SNPs)of 2 key genes:TRAF6(rs5030411,rs5030416),TLR4(rs1927914,rs10759932 and rs11536889).Statistical analyses were performed by SPSS17.0 for windows and the STATA11.0(STATA College Station,Texas,USA).Results:1.One sets of microarray data(GSE16561)were screened out from the gene chip database(Gene Expression omnibus,GEO)by using GSEA.One significant pathway(Toll-like receptor pathway)and its two key genes(TRAF6,TLR4)were acquired.2.The genotype frequencies of the TRAF6 gene variant(rs5030411,rs5030416),and the TLR4 gene variant(rs1927914,rs10759932 and rs11536889)in controls were consistent with HWE(P>0.05).3.Gene polymorphisms and ischemic stroke assiation analyses(1)TRAF6 gene polymorphisms of rs5030411 and rs5030416 were not statistically significantly associated with susceptibility of ischemic stroke under all four genetic models(all P>0.05).(2)No significant differences were observed in the association between TRAF6 gene polymorphisms of rs5030411 and rs5030416 and the susceptibility risk of ischemic stroke in male and female population.(3)TLR4 gene rs10759932 polymorphism was statistically significantly associated with susceptibility risk of ischemic stroke under the follow genetic models:the co-dominant 1 genetic model(Model 2:OR=0.095,95%CI:0.022-0.414,P=0.002),and the co-dominant 2 genetic model(Model 2:OR=0.130,95%CI:0.029-0.581,P=0.008),as well as the recessive genetic model(OR=9.120,95%CI:2.224-37.396,P=0.002).However,no significant association was found by rs11536889 polymorphism and susceptibility risk of ischemic stroke.(4)TLR4 gene polymorphism rs1927914 was significantly associated with the susceptibility of ischemic stroke:the co-dominant 1 genetic model(Model 2:OR=0.226,95%CI:0.067-0.764,P=0.017),while the recessive genetic model would increase the susceptibility risk of ischemic stroke(OR=3.522,95%CI:1.128-10.996,P=0.030).(5)TLR4 gene polymorphism rs10759932 was significantly associated with susceptibility of ischemic stroke in the female population:the co-dominant 1 genetic model(Model 2:OR=0.024,95%CI:0.002-0.364,P=0.007),and the co-dominant 2 genetic model(Model 2:OR=0.014,95%CI:0.001-0.234,P=0.003).In addition,we found that the rs10759932 polymorphism significant association with the susceptibility risk of ischemic stroke of the female population in the recessive genetic model(OR=52.29,95%CI:3.753-728.48,P=0.003).(6)TLR4 gene polymorphism rs1927914 was significantly associated with susceptibility of ischemic stroke in the female population:the co-dominant 1 genetic model(OR=0.070,95%CI:0.006-0.865,P=0.038),and the co-dominant 2 genetic model(OR=0.065,95%CI:0.007-0.630,P=0.018).In addition,we found that it polymorphism can increase the susceptibility risk of ischemic stroke in the female population of the recessive genetic model(OR=15.078,95%CI:1.621-140.25,P=0.017).(7)For the male population,we found the rs1927914 genetic variation can reduce the susceptibility risk of ischemic stroke(the dominant genetic model,OR:0.147,95%CI:0.023-0.952,P=0.044).4.The assiation analyses of gene polymorphisms and the susceptibility ischemic stroke with different TCM syndromes.(1)The Wind syndrome:No significant association were observed between TRAF6 gene polymorphisms of rs5030411and rs5030416 as well as the TLR4 gene polymorphism of rs11536889,rs10759932 and rs19279.14 with the susceptibility risk of ischemic stroke of Wind syndrome(all P>0.050).(2)The Phlegm syndrome:No significant association were observed between TRAF6 gene polymorphisms and the TLR4 gene polymorphisms with the susceptibility risk of ischemic stroke of Phlegm syndrome after corrected sex and age by Model 1 as well as adjusted multi confounding factors by Model 2 in ischemic stroke patients.(3)The Blood-stasis syndrome:No significant association were observed between TRAF6 gene polymorphisms of rs5030411and rs5030416 as well as the TLR4 gene polymorphism of rs11536889 and rs10759932 with the susceptibility risk of ischemic stroke of Blood-stasis syndrome(all P>0.050).While significant association was found between the TLR4 gene rs1927914 polymorphism and susceptibility risk of Blood-stasis syndrome in ischemic stroke patients:the dominant genetic model(OR= OR=2.182,95%CI:1.026-4.639,P=0.043).However,no significant association was observed between rs1927914 polymorphism and the susceptibility risk of ischemic stroke Blood-stasis syndrome after adjusted multi confounding factors by Model 2.(4)The Qi-deficiency syndrome:No significant association were observed between the TRAF6 gene polymorphism and ischemic stroke with Qi-deficiency syndrome(all P>0.050).In addition,no significant association were found between the TLR4 gene rs1927914,rsl0759932 and rs1927914 polymorphisms and susceptibility risk of Qi-deficiency syndrome in ischemic stroke patients.5.The TLR4 gene rs11536889 polymorphism was associcated with the serum low density lipoprotein level in ischemic stroke patients(P=0.046).Conclusions:(1)The Toll like receptor signaling pathway may be a key pathway for ischemic stroke.(2)TLR4 gene rs10759932 polymorphism may be associated with ischemic stroke in Han Chinese population,the genetic variation of A1A2 and A2A2 genotype may be a protective factor for ischemic stroke,they may reduce the susceptibility risk of ischemic stroke,especially in the female population.(3)TLR4 gene polymorphism of rs1927914 may be related to the susceptibility risk of ischemic stroke in Han population,the A1A2 and A2A2 genotype of it may reduce the susceptibility risk of ischemic stroke,especially in the female population.In addition,that association also can be verified in Han males population.(4)The TRAF6 and TLR4 gene may be not related to the susceptibility risk of Wind syndrome,Phlegm syndrome,Blood-stasis and Qi deficiency syndrome in ischemic stroke patients.(5)TLR4 gene rsll536889 polymorphism may be related to the metabolism of serum low-density lipoprotein in ischemic stroke patients.