Association Of-688G/C Single-nucleotide Polymorphism In The Promoter Region Of β-defensin1 Gene With Severe Sepsis

BackgroundSepsis represents a substantial health care burden.The current incidence of sepsis is at least 240 patients per 100,000 people in the United States population,whereas for severe sepsis it is between 51 and 95 patients per 100,000 people.The incidence rate for sepsis has been increasing over the past two decades,driving an increase in the number of deaths despite a decline in case-fatality rates.United States government had to spend more than 17 billion dollars per year on healthcare expenditures of septic patients.Sepsis,as defined by an expert consensus definition,is the development of the systemic inflammatory response syndrome in the presence of infection.The pathophysiological mechanism of sepsis and its sequelae severe sepsis,septic shock as well as multiple organ dysfunction(multiple organ failure)is severe systemic inflammation resulting from imbalance of proinflammatory and anti-inflammatory reaction.Sepsis is a multifactorial disease.It has great differences in susceptibility,severity and prognosis of individual who encounters sepsis,and impling inherited factors play a very important role in sepsis.Innate immune system plays an important role in sepsis and its sequelae.Defensins are small cationic peptides involved in innate immunity and are components of the first line of defence against invading pathogens.The human defensins can be subdivided into two main classes according to their structural differences:theα-defensins,β-defensins.β-defensin 1 was the first discovered humanβ-defensin.The spectrum of antimicrobial activity includes Gram positive and negative bacteria, fungi and enveloped viruses.It has been shown to be chemotactic for CD4⁺ memory T cells and immature but not mature dendritic cells.It has been demonstrated that in vitro,the expression ofβ-defensin 1 is increased in the present of LPS,IL-1β,INF-γor Arginine;in vivo,the concentration of plasmicβ-defensinl of septic or infected patients was more higher than controls.Furthermore,inter-individual difference in the level ofβ-defensin 1 has been discovered.HBD-1 gene(DEFB1 in HUGO/GDB nomenclature)was located at chromosomal region 8p23.1-p23.2 in close proximity(with in 100-150kb) to the gene for human neutrophilα-defensin HNP-1(DEFA1).The DEFB1 gene has no duplicons,but several single-nucleotide polymorphisms(SNPs)has been discovered in the gene.Previous studies have explored that single-nucleotide polymorphisms in the DEFB1 gene was associated with inflammatory and infected diseases,containing COPD,asthma,HIV-1 infection and atopic dermatitis.Sun CQ and his coworkers reported that a polymorphism at -688 bases upstream of the ATG start codon affects hBD-1 promoter activity,leading to a rate of reporter gene transcription that is 40%to 50%lower than the wild-type sequence when tested in either DU145 or TSU-Pr1 cell lines. Furthermore,they also found that there was a trend toward higher frequency of prostate cancer patients with at least one mutant base compared with controls.However,it has been never reported that the correlation between -688G/C SNP in DEFB 1 gene and severe sepsis.ObjectiveTo explore whether the singe-nucleotide polymorphism at the position -688 in the promoter region ofβ-defensin 1 gene(DEFB1)was associated with the incidence and outcome of severe sepsis.Materials and MethodsAfter approval by the hospital and national ethics committee, written informed consent was obtained from the patient or first degree relative.Two hundred and eleven postoperation patients were enrolled after diagnosis of severe sepsis according to the American college of Chest Physician/Society of Critical Care Medicine Consensus Conference Communittee criteria.Patients who were pregnant or whose age were less than 18 years were excluded.The severity of sepsis was evaluated by APACHEâ…¡and Sepsis-related Organ Failure Assessment Score. Meantime,one hundred and fifty-seven postoperative patients without sepsis were enrolled as controls.After acquired informed consent of patients,ten milliliter of peripheral whole blood was obtained from each individual through a veinous puncture(2%EDTA anticoagulation).Genomic DNA was isolated with QIAamp DNA Blood Mini Kit(QIAGEN Co.Ltd.)from 200uL whole blood.The genomic DNA was dissolved in 200uL TE,then conserved in -80℃.The primers for polymerase chain reaction(PCR) were designed based on the DNA sequence provided by Genbank(access numbers:NM₀05218,U50930),and synthesized by Shanghai BioSia Biotechnology Co.Ltd.The target gene was augmented through polymerase chain reaction(PCR)in Mastercycler gradient PCR instrument(Germany Eppendorf Co.Ltd.).Cac8I(New England BioLabs Co.Ltd)restriction endoenzyme digested the PCR products.The mixture was used for 1.8%agarose gel electrophoresis(ethidium bromide 0.125μg/ml).After electrophoresis,the stained genotypes were detected through ultraviolet image analysis.SPSS 13.0 statistics analysis software was used to analyzed the experimental data.Quantitative data was presented as mean±SD.Differences in genotype and allele frequencies between severe sepsis patients and healthy controls were assessed by x² test or Fisher's exact test.P value less than 0.05 was considered statistically significant.ResultThe variation of either the genotype frequency or the allelic frequency of DEFB1 -688 in patients with severe sepsis showed no significant difference with that in controls(P＞0.05).Furthermore,when the patients with severe sepsis were divided into survivors and non-survivors,there was also not significant difference in the distribution of genotype and allelic frequency between these two sub-groups (P＞0.05). ConclusionThe polymorphism at the position -688 in the promoter region of DEFB1 may not be associated with the susceptibility to and outcome of severe sepsis.The polymorphism at the position -688 in the promoter region of DEFB1 can not be to consider as a marker for assessment of occurrence and development of severe sepsis.