Genetic Susceptibility To Sepsis Based On The Candidate Gene Strategy

Background and Objective:The systemic response to infection has been termed sepsis which is a common complication after trauma,empyrosis,surgery and critical illness.Sepsis and its sequelae represent a continuum of clinical and pathophysiologic severity,including severe sepsis,septic shock,multiple organ dysfunction syndrome (MODS) and death.Epidemiologic survey has demonstrated that incidence of sepsis increases by nearly eighty times from approximately 0.3%in the 18-29 age decile to 24.2%in the 90-99 age decile.Stratified by age≥65 and＜65,the effects of age on sepsis mortality is independently associated with a 2.3 times higher risk of death.Men are more likely to develop sepsis compared to women and relative risk(RR) is 1.3. Incidence rate of blacks is nearly twice as high compared to whites and RR is 1.90.The mortality rate for blacks is also significantly higher than for other groups.By this token, genetic factors have a substantial impact on onset and outcome of sepsis.In 2001, SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference suggested that genetic factors should be taken as a part of causing mechanism of sepsis in further researching for its greater role in determining the risk of sepsis.Objective of our research is to identify the genetic polymorphisms associated with susceptibility to or severity of sepsis.Methods:Candidate genes for sepsis,which play a critical role in pathogenesis of sepsis,are screened and then,genetic variations across candidate genes are confirmed in considering all four factors,including association with other diseases,location in gene, linkage disequilibrium status and minor allele frequence.We extracted genomic DNA from 5 ml EDTA-anticoagulated peripheral blood samples using standard phenol/chloroform protocols and validated the minor allele frequence of SNP by sequencing after PCR amplification.The association of candidate polymorphisms with susceptibility to or severity of sepsis was analyzed in a case-control study consisted of 255 patients with sepsis and 260 volunteers from community as control.Then,GenomeLab SNPstream genotyping platform(Beckman Coulter Inc.) and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism(PCR-RFLP) method were employed to genotype.Unconditional logistic regression analysis was performed to analyze the association of polymorphisms with risk and severity of sepsis. P values,odds ratios(ORs),and 95%confidence intervals(CIs) were calculated.We adjusted five confounding factors,including age,sex,smoking,drinking and chronic diseases status in five genetic models for analysis of susceptibility to sepsis.In analyzing the severity of sepsis,two other confounding factors,APACHEⅡscoring and critical disease status,were also considered.Multiple testing corrections,including Bonferroni and False Discovery Rate(FDR),were used to correct error of typeⅠ.Results:The tests of association between SNP and risk of sepsis is performed firstly. Single SNP analying shows 13 SNPs of 8 genes are associated with susceptibility to sepsis in five genetic models,including tPA-rs2020922,rs2070713,F2-rs2070852, rs5896,SELP-rs3753306,rs2244529,rs3917647,SELE-rs4656700,rs2076059, NOS2A-rs2779248,C5-rs17216529,C5AR1-rs10404456 and TLR2-rs3804100. Haplotypes of five genes are associated with susceptibility to sepsis,including SELE, SELP,PAI-1,F2 and tPA.Allele statistic analysis displays allele frequencies of 9 SNPs of 6 genes are significantly different between case and control groups,including tPA-rs2020922,SELP-rs3753306,rs2244529,rs3917647,SELE-rs4656700, F2-rs2070852,rs5896,C5-rs17216529 and MBL2-rs2384044.Secondly,we analyze the association of SNP with severity of sepsis.Single SNP analying demonstrates 20 SNPs of 13 genes are associated with severity of sepsis, including SELE-rs2076059,SELP-rs3917647,NOS2A-rs2297518,F3-rs958587, F5-rs13306334,rs6020,rs6030,PAI-1-rs6092,tPA-rs2020922,rs2070711, F2-rs2070852,rs5896,C3-rs17030,rs2230205,rs428453,C5-rs10985126,rs12237774 and MBL2-rs2384044,TLR3-rs3775296andTLR5-rs2072493.Haplotypes of 4 genes are associated with severity of sepsis,including SELE,F3,F5 and C5.Allele statistic analysis finds allele frequencies of 7 SNPs in 5 genes are significantly different between different groups,including SELE-rs2076059,F3-rs3761955,rs958587,F5-rs13306334, rs6020,C3-rs2230205 and MBL2-rs2384044.Multiple testing corrections,including Bonferroni and FDR,are used to control the error of typeⅠ.After correction,frequency of allele A of rs2020922 is still higher in case group than control(P=7.0×10^-6).A allele can increase the probability of risk of sepsis (Log-additive,P＜0.0001),T/A genotype is associated with increased susceptibility to sepsis(P＜0.001) with the OR(95%CI) being 2.27(1.52-3.41) and A/A genotype is also associated with increased susceptibility to sepsis(P=0.042) with the OR(95%CI) being 5.30(1.06-26.36).In the dominant model,T/A-A/A combination genotype is associated with a two times higher risk of sepsis(P＜0.0001,OR=2.37,95%CI:1.59 -3.53).rs2076059 of SELE is associated with severity of sepsis in group which is composed of subjects with the number of organ dysfunction being more than two and not more than two.G/A genotype has significantly increased risk of MODS compared with the G/G genotype(P=0.002,OR=12.77,95%CI:2.49-65.40),G/A-A/A combination genotype is associated with a 12.79 times higher risk of number of organ dysfunction being more than two.(P=7.0×10^-4,OR=12.79,95%CI:2.49-65.57).Haplotypes of SELE,SELP and tPA are associated with susceptibility to sepsis. Subjects earring the G-G-A haplotype of SELE have significantly increased risk of sepsis compared with the G-C-G haplotype(P=0.0085).After multiple testing correction,P is still less than 0.05 and OR is 1.56 with 95%CI being 1.12-2.18.The possibility of incidence of sepsis is 1.97 times in subjects with C-C-A haplotype of tPA than C-T-T haplotype(P=0.0012,OR=1.97 and 95%CI:1.31-2.97).There is a significant difference in the level of global haplotype association for SELP(P=0.0024) after multiple testing correction,but all P values obtained from comparing each haplotype with the reference are more than 0.05 after correction.Conclusion:Allele A of rs2020922 of tPA gene can increase the risk of sepsis with dosage effect,and T/A and A/A genotypes can cause sepsis more easily than T/T genotype.G-G-A haplotype of SELE and C-C-A haplotype of tPA can increase the susceptibility to sepsis.G/A genotype or G/A-A/A combination genotype of rs2076059 of SELE can result in dysfunction of more than two organs.