Association Study Of TLR3,TLR4,TLR7 And TLR8 Gene Polymorphisms With The Severity Of EV71-associated Hand,foot And Mouth Disease

Background and ObjectiveHand,foot,and mouth disease（HFMD）is an infectious disease common in preschool children caused by enteroviruses,widely prevalent in the Asia-Pacific region and China ranks first in the world in terms of HFMD cases and mortality.In recent years,toll-like receptors（TLRs）have been found to be involved in the occurrence and development of HFMD.The present study aims to explore the association between TLR3,TLR4,TLR7 and TLR8 gene single nucleotide polymorphisms（SNPs）and the severity of HFMD caused by enterovirus 71（EV71）infection,and uncover the difference of clinical parameters between severe and mild patients and the influence of genetic factors on the clinical symptoms of EV71-associated HFMD patients.MethodsBased on the "Disease Surveillance Information Report Management System" of Anhui Province,328 children with HFMD caused by EV71 infection reported by various medical institutions from 2011 to 2018 were selected as subjects,of which 202 were male and 126 were female.They were divided into severe and mild groups according to the severity of the disease,with 176 in the severe group and 152 in the mild group.All subjects met the guidelines for diagnosis and treatment of HFMD（2010 version）and the patients were grouped based on the criterion.Snap Shot and first-generation sequencing were used to detect the polymorphic sites of TLRs genes（TLR3 gene: rs3775290,rs3775291,rs3775296,rs1879026,rs5743312,rs5743313,rs5743303,rs13126816,rs3775292;TLR4 gene: rs4986790,rs4986791,rs2149356,rs11536889,rs41426344;TLR7 gene: rs179009,rs179010,rs179016,rs3853839,rs2302267,rs1634323,rs5741880;TLR8 gene: rs3764880,rs2159377,rs2407992,rs5744080,rs3747414,rs3764879,rs5744069）and analyzed the association of genotype and allele frequency with the severity of EV71-associated HFMD.Meanwhile,the clinical information of 239 subjects were collected,and the differences in clinical parameters between the mild and severe groups and the influence of genetic factors on the clinical parameters of patients were analyzed.Results1.The individuals with TLR3 rs1879026 GG genotype were at higher risk of developing severe cases（GG vs GT: OR=1.875,95% CI,1.183-2.971,P=0.007）.The results of gender stratification showed that the CC genotype and C allele of TLR3rs3775290 were related to milder disease in women（CC vs CT: OR=0.350,95% CI,0.163-0.751,P=0.006;C vs T: OR=0.566,95% CI,0.332-0.965,P=0.036）.Female carriers of TLR3 rs3775291 CC genotype were 2.537 times more likely to develop severe cases than CT genotype（CC vs CT: OR=2.537,95% CI,1.108-5.806,P=0.026）.In addition,TLR3 rs1879026 GG genotype was associated with increased risk of severe disease in males（GG vs GT: OR=2.076,95% CI,1.144-3.768,P=0.016）.2.Haplotype analysis revealed three haplotype blocks（TLR3 Block 1: rs5743303,rs13126816 and rs3775296;TLR8 Block 1: rs3764879 and rs3764880;TLR8 Block 2:rs5744069,rs2159377,rs5744080,rs2407992 and rs3747414）.These SNPs constituted a total of nine haplotypes（TLR3 Block 1: AGC,AAC,TGA and AGA;TLR8 Block 1:GG and CA;TLR8 Block 2: TTTCA,GCCGC and TTCCA）.There was no statistically significant difference in haplotype distribution between the mild and severe groups.3.Severe cases and mild cases had significant differences in fever duration,blood glucose,platelet count and creatine kinase-MB（CK-MB）.Severe cases had higher fever duration（P=0.001）,blood glucose（P<0.001）,platelet count（P=0.004）and CK-MB concentration（P=0.003）.4.The three genotypes of rs1879026 and rs3775290 were not related to the clinical parameters of EV71-associated HFMD patients,but individuals with rs3775291 TT genotype had a higher alanine transaminase（ALT）concentration than those of CC and CT genotypes（P=0.037）.Conclusion1.The genetic variations of TLR3 rs1879026,rs3775290 and rs3775291 are related to the severity of EV71-associated HFMD in children.No association is found between the included TLR4,TLR7 and TLR8 polymorphisms and the severity of EV71-associated HFMD.Meanwhile,haplotype analysis does not identify haplotypes associated with HFMD severity2.Compared with mild patients,severe patients have higher fever duration,platelet count,blood glucose and CK-MB concentration.3.The rs3775291 TT genotype is associated with higher ALT concentration.