Association Of Human β Defensin 1 Gene Polymorphisms With Sepsis

Sepsis and its sequelae such as severe sepsis, septic shock and multiple organ dysfunction syndrome are a major cause of morbidity and mortality on today's intensive care units. The evidence that endogenous mediators actually mediate the individual's response to infection has led to various approaches to assess the individual's contribution to the course of the disease. The role of an individual's genetic background and predisposition for the extent of inflammatory responses is determined by variabilities of genes encoding endogenous mediators that constitute the pathways of inflammation. All genes encoding proteins involved in the transduction of inflammatory processes are candidate genes to determine the human genetic background that is responsible for interindividual differences in systemic inflammatory responses to infection. The gene determined the capacity of cytokine production and release, gene polymorphisms of coagulation factors or factors of the innate immune system-like defensins, and other genes involved in inflammation maycontribute to a wide range of clinical manifestations of an inflammatory disease. Genomic information may be used to identify groups of patients with a high risk of developing severe sepsis and multiple organ dysfunction.Innate immune system plays an important role in sepsis and its sequelae. Components of the innate immune repertoire of airway epithelia include human β -defensins (DEFB) and the only known human cathelicidin, LL-37. Human beta defensins ( DEFB) are cationic antimicrobial peptides playing important roles in immunoreactions not only with broad spectrum antibacterial, and antifungal as well as antiviral activities, but also chemotactic activity for dendritic cells and T cells. DEFB1 is mainly expressed in skin and other epithelial tissues all over the body. DEFB1 takes part in airway mucosal defence, and can effectively prevent microorganism infection. The gene encoding DEFB1 is located in region p23 of chromosome 8. Fifteen single nucleotide polymorphisms loci in DEFB 1 gene have been reported, of which seven exhibit differences in frequency among ethnic groups .It has been reported that a significant correlation between a single nucleotide polymorphism in the 5'-untranslated region of DEFB1 gene and the risk of HIV-1 infection in an Italian paediatric population. In another study, SNPs in this region are associated with asthma phenotypes and concentration of IgE in Chinese children. DEFB1 -44C/ G polymorphismis associated with oral Candida carriers in type 1 diabetics in American and show a correlation with COPD in Chinese Han population. DEFB1 1654G/ A polymorphism is a nonsynonymous SNP in exon 2 of DEFB1 gene. It has been reported that this mutation in COPD patients is prominent high than in control in Japanese.To present, there is no study investigating the relationship between genomic polymorphisms in DEFB 1 gene and susceptibility and outcome of sepsis.ObjectiveThe present study is to analyze the correlation between polymorphisms in DEFBl gene and the susceptibility and outcome of sepsis in Han Chinese population by case-control association analysis.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 sixty-eight postoperation patients were enrolled after diagnosis of sepsis according to American college of Chest Physician/Society of Critical Care Medicine Consensus Conference Communittee criteria. The severity of sepsis was evaluated by APACHE II and Sepsis-related Organ Failure Assessment Score. Oxygenation index was deem to the criteria of lung dysfunction. Meantime, one hundred and fifty-seven postoperative patients without sepsis wereenrolled as controls.Ten milliliter of peripheral whole blood were obtained from each individual through a veinous puncture1. Genomic DNA was isolated with QIAamp DNA Blood Mini Kit (QIAGEN .Co.) 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 fragment of DEFB1 5'-untranslated region including SNP -52A/G(rs 1799946) ,-44C/G(rs1800972)and -20A/G(rs 11362), and another fragment including SNP-1816A/G (rs2741136) were amplified using PCR. These four SNPs were analyzed by means of direct DNA sequencing. The results were confirmed by restriction fragment length polymorphism (RFLP) analysis (random 30 samples).SNP -390A/T ( rs2738182 ) was measured with method of Allele-specific PCR amplifications (PCR-ASA). Tow sense primers were designed for amplification of the mutation type and wild type, respectively. 3'extremity of tow primers were exactly match the allele A and T. Two antisense primers were not identical. Every sample was amplified the fragments by two couples of primers respectively. Four microliter of PCR product from two different amplifications was mixedcompletely. The mixture was used for 2 % agarose gel electrophoresis (ethidium bromide 0.125 μg/ ml). After electrophoresis, the stained genotypes were detected through ultraviolet image analysis: A/A genotype referred to a 408 bp band , T/T genotype referred to a 263 bands , and A/T genotype referred to two bands (408 bp and 263 bp ). The results were confirmed by direct DNA sequence (random 30 samples).SNP 1654G/A was analyzed by high-throughput site-specific TaqMan assay. The primers, probes, and assay conditions were provided from SangHai GenKone Co. The 7900 sequence detection system (ABI.Co), a 96-well plate, laser and fiber optic reading instrument, was used for high-throughput analysis of genomic DNA samples. Fluorophores VIC, FAM and a quencher TAMRA were incorporated into the specific wild-type FAM and mutant VIC probes for SNP determination.All single-nucleotide data were evaluated for Hardy-Weinberg equilibrium. Differences in genotype and allele frequencies between sepsis patients and healthy controls were assessed by x² test or Fisher's exact test . The intensity of association was estimated by the odds ratio (OR) and 95%CI. Differences in APACHE II , SOFA and OI among different genetypes were evaluated by Kruskal Wallis test. P value less than 0.05 was considered statistically significant.Result1 The ratio of men to women for the sepsis population was 165:103, and that for the controls was 96:61. The mean age of both groups was comparable (60.0±17.9 VS 58.4±18.3). No significant differences in age and sex construction between sepsis patients and controls.2 All SNPs were in Hardy-Weinberg equilibrium. (P>0.05)3 One hundred and ninety-three CC homozygote ( 72.0 %) , 65 CG heterozygote (24.2 %) and 10 GG homozygote (3.8 %) genotypes for -44C/ G locus were detected from the 268 sepsis patients from which the frequencies of C and G alleles were 84.1 % and 15.9 % , respectively. In the 94 controls, 130 CC homozygote (82.8 %), 27 CG heterozygote (17.2 %) and 0 GG homozygote genotypes were detected and the frequencies of C and G alleles were 91.4 % and 8.6 %, respectively. The distribution of genotype frequency and allele frequency in sepsis was significantly different from that in controls(P<0.05), the OR value is 2.00(1.27-3.17). In another SNP -20A/G (rs11362), the G allele frequency in sepsis patients was significantly higher than that in controls(P<0.05), the OR value is 1.47(1.11-1.96). There were no significant differences in distribution of genotype and allele frequency between sepsis patients and controls for other SNP. The 1654A/G SNP was not found in these study..4 In septic patients, the distribution of genotype and allele frequency inSNP -44C/G and -1816A/G were significantly different between survivor and non-survivor. 5 Oxygenation index was significantly lower in sepsis patients with thegenetype GG homozygote at -1816A/G than with the other genetypes.But there was no significant differences in APACHE II andSepsis-related Organ Failure Assessment Score .ConclusionTo our knowledge, this is the first study that reports a significant association between SNP in the DEFB1 gene and sepsis in Han Chinese. In this study , novel genetic variation in DEFB1 showed a positive association with sepsis (-44C/G and -20A/G were associated with the susceptibility of sepsis, -44C/G and -1816A/G were associated with the outcome of sepsis, in addition, the GG homozygote in -1816A/G was associated with the severity of lung dysfunction) Although the positive SNP may be only one of several factors that contribute to susceptibility and outcome of sepsis, it could prove to be a valuable marker for assessment of risk for sepsis.