Study Of Relationship Between The Polymorphisms Of TLR9and EBV Infection In182Children

Objective: Toll-like receptor9(TLR9) is a kind of natural pattern recognitionreceptors expressed on the cell surface, it plays an important role in the immuneresponse. In this study, according to detect4different SNPs which located in thepromoter, intron and exon of TLR9gene, we analyze the relationship between thepolymorphism of TLR9and the EBV infection in children from the genetic level andtry to find the reason why the clinical type and the outcome after EBV infection aredifferent in different children.Methods: Collected the data of clinical cases and peripheral venous blood,there are3cases with EBV infection-associated hemophagocytic syndrome,56caseswith infectious mononucleosis,69cases with EBV active infection but with mildclinical symptoms, and54cases without EBV infection (EBV-antibodies werenegative). We finally selected rs187084(T/C), rs5743836(T/C) which located inpromoter region, the rs352139(A/G) located in the intron and rs352140(G/A)located in the exon region of TLR9gene as the object of study by consulting theinformation of SNPs in databases and combining with related literatures. GenomicDNA was extracted from peripheral blood by salting out method. Primers weredesigned using Primer5, Oligo2and NCBI database, then Polymease Chain Reaction(PCR) was used to amplify the DNA fragments which contained the selected SNPloci. PCR products were sequenced directly, and we analyzed the genotype of SNPsaccording to the peak figures of sequencing results to find the differences ofgenotype and allele frequency among different groups.Results:(1)For the people in this experiment, there are three genotypes ofrs187084: TT, TC and CC, three genotypes of rs352139: AA, AG and GG, threegenotypes of rs352140: GG, GA and AA, and the MAF is39.7%,38.8%and41.9%respectively. The above three sites with polymorphisms in the experimental populations and their distribution are similar to the situation of Chinese populationpublished in the NCBI (dbSNP) database and Ensembl (1000Genomes) database.But there are only two genotypes of rs5743836: TT and TC, while TC genotype wasfound only one case in all samples, indicating that there is no obvious polymorphismabout this locus in the experimental populations.(2)There are3cases aboutEBV-HLH, the genotype of each SNP as follows: rs187084: T/T in2cases and1case of T/C; rs352139: A/A in2cases and1case of A/G; rs352140: G/G in2casesand1case of G/A.(3)Among other groups, the genotype and allele distribution ofeach SNP are shown as follow:Group1: the group of IM. Group2: the group of patients with mild clinical symptomsof EBV active infection. Group3: the control group of EBV-negative infection.There was no statistically significant difference about the genotype and allelefrequency of all the selected SNPs among the three groups (P>0.05).Conclusion:(1)The salting out method is a simple method to extract DNA fromperipheral blood while there are many samples, the DNA concentration and puritywere both ideal.(2) There is no polymorphism about the rs5743836of TLR9geneSNP in the182cases of children, it makes little sense to test this site genotype whendo the related research.(3)The rs187084, rs352139and rs352140of TLR9gene areall with polymorphism in the study population, and the distribution is similar to thesituation published in the related database. We did not find there are correlationbetween the polymorphism of these SNPs and EBV infection in children.