Association Of Some Key Single Nucleotide Polymorphisms With Risk Of Development Of Complications In Patients With Major Trauma

Background:Major trauma is the leading cause of death in young adults and the third most common cause of death overall. Victims of severe injuries who survive the initial hours are likely to develop sepsis and sepsis-associated multiple organ dysfunction syndrome (MODS), which remains a worldwide problem that is the leading cause of intensive care unit mortality. The pathogenesis underlying sepsis and MODS has been demonstrated to be overwhelming immune inflammatory response, which might be preventable and controllable if being effectively managed at the early stage. To this end, it might be pivotal to find an effective way for evaluation of the risk for the sepsis and MODS in major trauma patients.Cytokines are critical determinants for the magnitude of immune inflammatory response, which might profoundly affect the inflammatory response to trauma and predispose trauma patients to susceptibility or resistance to sepsis and MODS. While pattern recognition receptors (PRRs), which have been shown to be elevated after injury or hemorrhagic shock, are considered as important mechanism for that injury primes the innate immune system and leads to overwhelming proinflammatory response when bacteria and their products enter into body. Therefore, SNPs of some important cytokines and PRRs were selected. Their functionality and clinical relevance were analyzed through bioinformatic studies, in vitro functional studies and clinical association studies.Materials and Methods:1. Study population. A total of 656 unrelated adult Chinese, comprising 348 healthy blood donors and 308 patients with major trauma, were recruited in this study. All of them are Han Chinese and live in the Chongqing district. The 348 healthy blood donors consisted of 219 men and 129 women, with a median age of 25 yrs (range, 18–53 yrs). The 308 trauma patients, 242 men and 46 women, were consecutively admitted to the Department of Trauma Surgery in the Daping Hospital and the Chongqing Emergency Medical Center, Chongqing, China, between January 1, 2005, and January 1, 2008. They were enrolled in the study if they met the following criteria: 1) between 16 and 70 yrs of age, 2) expected Injury Severity Score of 16, and 3) probability of survival of 1 wk (sepsis or multiple organ dysfunction usually occur 1 wk after trauma). Their average age was 38.5 and median ISS was 25.5.2. Sequencing of the IL-6 Promoter and bioinformatic analysis. According to distribution of IL-6 promoter SNPs in the International HapMap Project SNP database, a 3-kb sequence from position -2996 to +53 of the IL-6 gene was sequenced for discovery of SNPs (refer to GenBank, Accession No. Y00081.1). Human genetic DNA samples were collected from 27 unrelated, healthy Han Chinese subjects. Overlapping primer sets were designed on the basis of size and overlap of polymerase chain reaction amplicons by use of Primer 3.0. SNPs were found through conting construction and multiple assemble. Then, IL-6 -572C/G was analyzed using online bioinformatics tools (http://125.itba.mi.cnr. it/genebin/wwwrepeat.pl and http://motif.genome.ad.jp/) to predict its potential functions.3. Report assay. In order to further validate the effect of -572C/G on IL-6 promoter activities, the wildtype IL-6 gene promoter, a sequence of -1728~+36bp, was amplificated. pGL3BH-CC plasmid was constructed and Site-Directed mutated to pGL3BH-GG plasmid. After transfection of the both plasmids into U-937 and K-562 cell lines, luciferase activity was measured using the Luciferase Assay System.4. Preparation of allele-specific oligonucleotide array. Allele-specific oligonucleotides(ASOs) were designed with Oligo 6 and synthesized using an Expedite 8909 nucleic acid synthesizer with a 5-terminal (CH2)6–NH2 modification and purified by perfusion chromatography on a BioCAD Sprint system. The ASOs were diluted to 100μmol/L with 3×SSC and were spotted on aldehyde coated glass slides using a Cartesian Pixsys 7500. The slides were stored at -4°C until use. Before use, the slides were washed in 0.2% SDS for 3 min and pure water for 2 min, and dried in the air.5. Genotyping. Genotyping was performed through RFLP-PCR (CD14/-159, -1145, MD-2/-1625 and IL-6/-572) and SNP array (13 cytokine gene polymorphisms) respectively. The results were confirmed by resequencing random ten samples. 6. Plasma cytokines'level assay. The whole blood collected from the healthy blood donors and trauma patients within 24 hrs after admission were mixed 1:1 with Roswell Park Memorial Institute 1640 culture medium, and incubated with 100ng/ml lipopolysaccharide in a sample mixer at 37°C for 4 hrs. The cytokines'levels in the supernatants were determined with enzyme-linked immunosorbent assay according to the manufacturer's instructions.7. Clinical evaluation. The patients with major trauma were prospectively monitored after admission by physicians who did not know the genotypes. Sepsis was defined if patients met all the following criteria: clinical evidence of infection, body temperature of 38.5°C or 36.5°C, and leukocyte count of 10×109/L or 4×109/L. When patients were diagnosed with sepsis, assessments of respiratory (PaO2/FIO2 ratio), hepatic (serum bilirubin), renal (serum creatinine), cardiovascular (pressure-adjusted heart rate), hematologic (platelet count), and central nervous (Glasgow Coma Scale) systems were made and calculated multiple organ dysfunction scores. ISS was performed according to the abbreviated injury scale 1998.8. Statistical analysis. Allele frequencies for each SNP were determined by gene counting. Genotype distribution was tested for departure from Hardy-Weinberg equilibrium using chisquare analyses. Luciferase activities were compared using one-way analysis of variance. The association between SNPs and MODS or plasma cytokines'levels was determined using one-way analysis of variance. We performed linear regression analysis to quantify the allele-dose effect. The association of genotypes with sepsis morbidity was determined by chi-square analysis. Odds ratios with 95% confidence intervals were calculated by multiple logistic regression analyses to estimate the relative risk of sepsis. The power was calculated by PS: Power and Sample Size Calculation software (http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize). Results were considered to be significant at P<0.05. All statistical analyses were carried out using SPSS Version 11.0. Results:1. Only one variant (C-572G) was identified in IL-6 promoter in Chinese Han population, with a frequency of 27.8%. The C/G variation at position -572 could reduce transcriptional activity of the IL-6 promoter as shown in both U-937 and K-562 cell lines and was associated with IL-6 production by peripheral leukocytes in response to ex vivo lipopolysaccharide stimulation in an allele dose–dependent effect. Moreover, the -572 polymorphism was associated with lower risk of sepsis in major trauma patients.2. Nine key cytokine genes and 13 SNPs (IL-1α-889C/T,IL-1β-1470G/C,-511C/T,-31C/T,IL-4 -589T/C,IL-6 -572C/G,IL-8 -251T/A,IL-10 -1082A/G,-819T/C,-592A/C,TNF-α-308G/A , TNF-β252G/A , IFN-γ874A/T) were selected. Allele-specific oligonucleotide array was developed successfully and used for genotyping in 308 patients with major trauma. 8 SNPs (IL-1β-1470G/C,-511C/T,-31C/T, IL-4 -589T/C, IL-6 -572C/G, IL-8 -252T/A, IL-10 -819T/C, TNF-α-308G/A ) were selected out of the 13 SNPs according to their significant relationship with respective cytokine levels, sepsis morbidity and MODS scores. The number of hyper-responsive alleles of the 8 SNPs a patient has is significantly correlated with his risk of developing sepsis and MODS.3. Trauma patients carrying CD14/–1145 A or–159 C allele had lower multiple organ dysfunction score and sepsis morbidity rate when compared with those with–1145 G or–159 T allele. In addition, both polymorphisms were in strong linkage disequilibrium, and had a marked synergistic effect. While patients who possessed the MD-2/1625 G allele were more likely to experience complications with organ dysfunction and sepsis after major trauma.Conclusions:1. -572C/G is the only SNP found in IL-6 gene promoter in Chinese Han population. -597 and -174 poymorphisms, common SNPs in Caucasian population, are not exit in Chinese Han population. IL-6/-572C→G variation could reduce promoter activity and inhibit IL-6 gene transcription, showing a functional SNP.2. Allele-specific oligonucleotide array was successfully developed for genotyping 13 SNPs in 9 cytokine genes.3. Using SNP array, 8 SNPs out of the 13 SNPs (IL-1β/-1470G, IL-1β/-511C, IL-1β/-31T, IL-4/-589C, IL-6/-572C, IL-8/-252T, IL-10/-819T and TNFα/-308A) are shown to be well associated with the development of sepsis and MODS in trauma patients.4. CD14-159T/C, -1145G/A and MD-2 -1625C/G might be used as risk determinants for sepsis and MODS in trauma patients.