Identification Of Tag Single Nucleotide Polymorphisms Of Some Key Genes And Their Association With The Risk Of Development Of Complications In Patients With Major Trauma In Multicenter Studies

Background:Trauma, a major public health problem worldwide, ranks as the fourth leading cause of death among all diseases. One of the most serious complications of major trauma is the sequential dysfunction of vital organs, which is associated with post-traumatic sepsis in the majority of cases. Therefore, preventing sepsis and subsequent organ dysfunction is crucial in the treatment of surviving patients with major trauma. It has been demonstrated that dysbalanced immune inflammatory response contributes to the development of sepsis and MODS in major trauma patients. Increasing evidence suggests that genetic variants, particularly single nucleotide polymorphisms （SNPs）, are critical determinants for inter-individual differences in both inflammatory responses and clinical outcome in trauma patients. Delineating the variation in genes and associated differences in response to trauma may contribute to the development of new genetically tailored diagnostic and therapeutic interventions that will improve outcome in the patients with major trauma. Cytokines such as interlukin-10 （IL-10） 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. LBP and MD2 are important PRRs. LBP is not only essential in lipopolysaccharide （LPS） recognition and signaling in Gram-negative infection, but also has a central role in Gram-positive infection. High-mobility group box-1 （HMGB1） and its receptor for advanced glycation end-products （RGAE） are also important genes in the pathogenesis of sepsis. 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. Patients with major trauma recruited in this study were Han Chinese from Chongqing in south-western China and Zhejiang in eastern China. The trauma patients were admitted to the Department of Trauma Surgery in the Daping Hospital and the Chongqing Emergency Medical Center between January 1, 2005 and October 1, 2010, and to the Department of Trauma and Emergency in the Second Affiliated Hospital, Zhejiang University between January 1, 2007 and July 1, 2010. They were enrolled in the study if they met the following criteria: 1) between 18 and 65 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 week after trauma）.2. Haplotype-tagSNPs selection. 3-10 kb upstream of the transcription start site and 3-10 kb downstream of the stop codon of LBP, HMGB1, RGAE and MD-2 were observed in the current study.Genetic variation data for entire genes were obtained from the HapMap project for 45 healthy Chinese Han Beijing （CHB） population （www.hapmap.org）. Haplotype blocks were constructed using Haploview. The criteria for the selected SNPs to construct a haplotype block is that all SNPs in one region must be in strong LD with D’> 0.98 for the upper 95% confidence bound, and >0.7 for the lower bound. A maximally informative htSNP was then selected from each block using software Tagger program.3. Genotyping by Pyrosequencing. Pyrosequencing is a rapid and reliable method used for SNP haplotyping. The genotypes of the PCR products were determined by pyrosequencing analysis using a standard protocol. The reaction was carried out at 28°C. Each base of the various SNPs was compared with a reference counterpart located elsewhere in the sequence context, and a ratio was calculated. Genotyping was performed in a blinded fashion without knowledge of the patients’clinical data, and 10% of the samples were genotyped in duplicate to monitor genotyping quality.4. 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 100 ng/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.5. Molecular Modeling. Using the crystal structure of bactericidal/permeability increasing protein （BPI） （PDB code: 1ewf） as template, the 3-D theoretical structure of LBP was modeled based on computer-guided homology modeling method. The LBP and BPI protein sequences were initially aligned with Blastp method. Using distance geometry method, the distances of amino acids, the area and volume of the hydrophobic pocket were analyzed. The van der Waals energy was calculated by Discover₃ module.6. Preparation of recombinant wild and mutant human LBP. The full-length cDNA of the LBP gene was created by polymerase chain reaction amplification of human hepatic RNA. The LBP gene was inserted into the pCI-neo plasmid through a EcoRI site and a XbaI site. The 26877T→C mutation encoding the Phe436Leu change was introduced by site-directed mutagenesis.The plasmid constructs were verified by direct sequencing analysis. To prepare recombinant human LBP proteins, CHO cells were cultured. The CHO cells were transfected with the wild-type and mutated plasmids. The supernatants were then collected after 48 hours. The recombinant human LBP contents in supernatants were detected with ELISA.7. Effect of the rs2232618 polymorphism on the binding and activation of LPS. Raw264.7 cells were cultured. After confluence, the cells were treated with serum-free RPMI 1640 medium containing 200 ng/ml of wild-type or mutant recombinant human LBP protein and 100 ng/ml LPS or FITC-LPS for 2 hours. The supernatants were collected for assay of TNF-α. After thorough washing with PBS, the cells were used to detect fluorescence intensity with a spectrofluorimeter.8. Effect of the rs2232618 polymorphism on the binding of LBP to CD14. ELISA was performed to check the binding activity of wild or mutant recombinant human LBP protein with CD14. The 96-well microtiter plates were coated with 10μg/ml CD14. After washing 5 times with PBS, 100 ng/ml wild or mutant recombinant human LBP was added to each well and incubated for 1 h at 37°C. The LBP bound to CD14 was detected using goat anti-human LBP polyclonal antibody and HRP-conjugated rabbit anti-goat monoclonal antibody. The OD of each well was determined using an ELISA reader.9. The effect of rs1800625 （-429T/C） on RAGE promoter activity. The possible effect of -429T and -429C on the promoter activity was investigated using a reporter gene assay system. Two plasmid constructs were prepared by inserting a 1765-bp sequence （-597 +26） of the RAGE gene into a promoterless pGL3-Basic vector, which contained wild genotype of–429T and -429C. The constructed vectors were transiently transfected into Human U937 cells. At 24 h post-transfection, the cells were treated with LPS （100ng/ml） for 24 h. Luciferase activities were measured with a Luminoskan Ascent luminometer.10. 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 1998.11. 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. The -1082 polymorphism of IL-10 was closely associated with the development of sepsis, showing that the patients with the A allele had significantly higher sepsis morbidity. The -1082A and -592A alleles and ATA haplotype were significantly associated with lower IL-10 production in response to ex vivo lipopolysaccharide stimulation.2. The rs2232618 （Phe436Leu） of LBP was closely associated with the development of sepsis and MODs after major trauma in both Chongqing and Zhejiang cohort, showing that the patients with the C allele had significantly higher sepsis morbidity. The Phe436 was shown to be a highly conserved amino acid among different species. Its substitution with leucine causes spatial conformational changed of the active center of the C-terminal domain of the LBP protein, as shown by molecular modeling analysis. Our results from in vitro experiments revealed that the delivery of LPS to CD14 on the surface of macrophages and LPS-induced cellular activation, were more greatly enhanced with the presence of the variant 436Leu LBP protein than those with the presence of the wild-type Phe436 LBP protein. In addition, the variant 436Leu LBP protein also had a stronger binding interaction with CD14, as shown by ELISA assay.3. Among the three tag SNPs of HMGB1, only the rs2249825 polymorphism was shown to be well associated with LPS-induced HMGB1 production. The C to G variation of the rs2249825 polymorphism, though locating in the intron 1, might create a binding site for v-Myb （1）, which is shown to be a powerful enhancer of HMGB1 expression. In parallel to their association with LPS-induced HMGB1 production, rs2249825 polymorphism was associated with higher risk of sepsis and MODS in major trauma patients while the other two tag SNPs （rs1412125, rs1045411） did not affect the outcome of major trauma patients.4. Among the three htSNPs of MD-2, only the rs11465996 polymorphism revealed a strong clinical relevance in both Chongqing and Zhejiang cohort, showing higher sepsis morbidity rate and MOD scores and a stronger immune inflammatory response in patients with the variant G allele. Our results were in accordance with our previous studies （2）.5.The rs1800625 （-429T/C） of RAGE was showing a significant association with sepsis morbidity rate and MOD scores in major trauma patients. Patients who carried T alleles had a higher sepsis morbidity rate and MODs scores. The promoter luciferase activity further confirmed the result that -429C might decrease the activity of the RAGE promoter. Conclusions:The -1082 polymorphism of IL-10, rs2232618 of LBP, the rs2249825 of HMGB1, the rs11465996 of MD-2 and the rs1800625 of RAGE were all shown to be well associated with the development of sepsis and MODS in trauma patients. The subsititution of LBP/rs2232618 （Phe436Leu） might change the binding effect of LBP protein and CD14, causing changes of the binding and activation effect of LBP and LPS. RAGE/-429T→C variation could reduce the promoter activity of RAGE gene, showing a functional SNP. These polymorphisms might be used as risk determinants for sepsis and MODS in trauma patients.