A Haplotype Of Single Nucleotide Polymorphisms In Human β-Defensin-1 Gene Is Associated With Incidence And Outcome Of Sepsis

Sepsis is a systemic inflammatory response syndrome which is caused by causative microbes invading organisms. The imbalance between inflammation and anti-inflammation leads to its pathophysiological process. Severe sepsis, septic shock and MODS is its consequent syndromes. In USA, there are approximately 7.5 milliom patients who are involved in severe sepsis, and 2.15 million patients died in severe sepsis and its sequelae every year. The average mortality rate is 28.6%, while the mortality rate of old patients over 85 is to 38.4%. The care of patients with sepsis results in an economic burden of nearly ＄17 billion annually. It is the 10th leading cause of death overall in the United States. In China, with the development of economy and technology, particularly the application of the advanced medical technology such as invasive monitor and the irregularly using of antibiotics and immunosuppressant, in addition to the increasing old people, the currency of sepsis in china is comparable to that in the United States. Therefore, investigation of the mechanism of sepsis in order to improve treatmentand decline mortality is very important.In 1996, Professor Stuber first reported an association study between genetic variability and sepsis, and found that a genomic polymorphism within the tumor necrosis factor locus influenced plasma concentrations of tumor necrosis factor-alpha as well as outcome of patients with severe sepsis. Since then, researches about genetic variability in susceptibility and outcome of sepsis are gradually increased. At present, researchers have revealed that genomic varabilities in locus of TNF, Toll like receptors , PAI-1 and other factors are associated with susceptibility and outcome of sepsis. Defensin is one of the most important family of antimicrobial peptides and plays an important role in innate and adaptive immunity. Previous studies showed that genomic polymorphisms in human β-Defensin1 (HBD-1) gene are associated with some of infection or infection related diseases such as COPD and HIV. With the HapMap project going on, haplotype analysis becomes a powerful method to reveal the disease associated single nucleotide polymorphisms (SNPs). The purpose of present study is to identify SNPs in HBD-1 gene in Han Chinese population, further compare the haplotype pattern to analyze the relationship between the haplotypes and the susceptibility and outcome of sepsis.ObjectiveInvestigate single nucleotide polymorphisms (SNPs) and the distribution of their haplotypes in HBD-1 gene In Han people. Analyze the relationship between the haplotypes and susceptibility and outcome of sepsis.MethodsSelect the SNP sites which may influence the efficiency of transcription and translation of HBD-1 gene as well as change the amino acid sequence and subsequent protein structure or function: -1816A/G (rs2741136) ,-390A/T (rs2738182) located in the promoter region, -52A/G (rsl799946),-44C/G (rsl800972),-20A/G (rs11362) located in the 5' untranslated region (UTR), a nonsynonymous SNP 1654G/A (rs2738047) located in the exon 2 region. After extraction of genomic DNA from peripheral blood of 268 sepsis patients and 167 elective postoperative patients who didn't involve in sepsis, DNA sequence analysis was performed using three different analytic methods. The genotypes of -1816A/G, -52A/G, -44C/G and -20A/G were determined by polymerase chain reaction and direct sequencing (MegaBAC1000 sequencing kit). The genotypes of -390A/T were determined by PCR-ASA,and the genotypes of 1654G/A were determined by a TaqMan assay. Genotype frequency and allele frequency were calculated. A Haploview progress was used to analysis haplotype pattern and lingkagedisequilibrim. The significance of differences in allelic,genotype and haplotype frequencies was calculated by chi-square Fisher's exact test. P ≤ 0.05 was considered to be significantly difference. The statistical analysis were carried out using SPSS software (version 11.5; spss, inc., Chicago, IL, USA).Results1. Single nucleotide polymorphisms, genotype frequency and allele frequency in HBD-1 geneAll the genotypes of 1654G/A in the exon 2 region were GG homozygous. All the other five SNPs located in the promoter region and 5' untranslated region (UTR) have three kinds of genotypes. Genotype frequencies for both sepsis group and control group were in Hardy-Weinberg equilibrium (P > 0. 05). The differences between sepsis group and control group in allelic and genotype frequencies of -20A/G, -44C/G site were significant (P<0.05). The differences between non-survivors and survivors in allelic and genotype frequencies of -44C/G,-1816A/G site were significant (P< 0.05).2. Linkage disequilibrium analysisIn sepsis group, strong linkage equilibrium was observed between -20A/G and the SNP sites in the promoter region, -52A/G and -390A/T, -44C/G and -1816A/G, between the three SNP sites in the 5' UTR were also in linkage quilibrium. D'values were more than 0.7. Linkagedisequilibrium between the other SNP sites was tender or to linkage equilibrium. Linkage quilibrium between sepsis group and control group was similar. Linkage quilibrium between non-survivors group and survivors group was also similar.3. Haplotype analysis in Han peopleA total of 7 common haplotypes were estimated within the five SNP sites in HBD-1 gene in control group. Of them H1 and H2 were the majority haplotypes.4. The association between haplotypes and sepsisThe same haplotypes were observed in sepsis group, non-survivors group and survivors group too. The frequency of — 20G / — 44G / — 52G / —390T / — 1816G was significantly higher in sepsis group than control group( P <0.05). It was also significantly higher in non-survivors group than in survivors group( P < 0.05).ConclusionThere are seven common haplotypes in HBD-1 gene in Han people in China. Of them, -20G / -44G / -52G / -390T / -1816G is associated with susceptibility and outcome of sepsis.