Identification Of Haplotype Tag SNPs Within The Nuclear Factor-κB Family Genes And Their Clinical Relevance In Patients With Major Trauma

BackgroundTrauma accounts for 10% of deaths and 16% of disabilities worldwide. Because of advances in the emergency care system, such as the use of antibiotic prophylaxis, patients who experience severe trauma are now able to survive, while it is often complicated with sepsis and multiple organ dysfunction syndrome(MODS) [1]. For reasons that will be discussed subsequently, the control of sepsis and MODS in earlier time is crucial in the treatment of trauma patients. Recent laboratory research has improved our understanding of the interaction between imbalance of proinflammatory and anti-inflammatory factors and sepsis [2]. Owing to our increased understanding, the pathway between single nucleotide polymorphisms(SNPs) and body’s immunity against infection can be mapped in increasing detail [3, 4]. The results of these trials will not only be pragmatically useful, but will also enable a more complete understanding of the biological mechanisms of posttraumatic sepsis and MODS.Nuclear factor-κB(NF-κB) family plays an important role in the development of sepsis in critically ill patients. Although several single nucleotide polymorphisms(SNPs) have been identified in the NF-κB family genes, only a few SNPs have been studied. The human NF-κB family contains five members: p50, p52, p65(Rel A), Rel B, and c-Rel, which encoded by NFKB1, NFKB2, RELA, RELB and REL genes, respectively. These subunits form homo- and heterodimers that control a broad spectrum of biological processes, including development, apoptosis, and the immune response [5, 6]. In the resting cell, most NF-κB dimers are found in the cytoplasm in their inactive forms by binding to inhibitory proteins. Many extracellular stimuli, including bacterial lipopolysaccharide, viruses, oxidants, inflammatory cytokines, and immune stimuli, can activate NF-κB [7, 8]. Once activated, NF-κB dimers are released and translocate to the nucleus where they coordinate the transcriptional activation of target genes, such as those encoding proinflammatory cytokines, chemokines, enzymes relevant for inflammation, and adhesion molecules [9, 10]. NF-κB family regulates genes required for both the innate and adaptive immune responses [11, 12]. The pivotal role of NF-κB family in the development of sepsis makes it an interesting candidate for genetic analysis.NFKB polymorphisms may be associated with magnitude of proinflammatory response, thereby affecting susceptibility to acute and chronic inflammatory diseases [13]. To date, Adamzik [14] and Schafer [15] had reported that the deletion allele of the NFKB1 insertion/deletion(-94ins/del ATTG) polymorphism was associated with increased 30-day mortality in patients with severe sepsis and septic shock. However, there is little information about the clinical relevance of SNPs within NF-κB family genes in patients with sepsis.Objective of our research is to identify haplotype tag single-nucleotide polymorphisms(ht SNPs) within the nuclear factor-κB(NFKB) family genes and to investigate their clinical relevance in patients with major trauma.Methods1. The full sequence of the human NFKB1, NFKB2, RELA, RELB and REL genes observed in the current study included 5 kb up-stream of the transcription start site, all exons and introns and 5 kb downstream of the stop codon(www.ncbi.nlm.nih.gov/genbank/). Genetic variation data for the entire NFKB1, NFKB2, RELA, RELB and REL genes and their surrounding selected regions were obtained from the Hap Map project(www.hapmap.org) for 137 Han Chinese individuals from Beijing(CHB). Haplotype blocks were constructed using Haploview, version 4.2(Broad Institute of MIT and Harvard, Cambridge, Mass), a software package that provides computation of linkage disequilibrium(LD) statistics and population haplotype patterns from genotype data. Software Targetscan was used to analyze the function of SNPs selected from the 5’-flanking region of the NF-κB family genes.2. Tag SNPs within the canonical NF-κB pathway(NFKB1, RELA, and REL) were selected for genotyping in trauma patients.Study population. We identified 753 trauma patients between Jan 1, 2005 and Jun 1, 2014.Genotyping of tag SNPs. Blood specimens were collected in tripotassium ethylenediaminetetraacetic acid sterile tubes from trauma patients immediately after admission to avoid the effect of blood transfusion. The genomic DNA was isolated from whole blood according to the manufacturer’s protocol. DNA concentration in all samples was determined by ultraviolet spectrophotometry, adjusted to a concentration of 40 μg/m L with sterile distilled water and stored at-80?C. Pyrosequencing was used for genotyping [16].Statistical analysis. We chose the log-additive inheritance model, which is the most suitable one for polygenic diseases. We did allele frequencies analyses by calculating. We assessed genotype distribution with Hardy-Weinberg equilibrium(HWE) by χ2 analyses. We assessed the relation of SNPs and MOD scores using covariance test. The extent of pair-wise linkage disequilibrium(r2-value) between polymorphisms was determined by the Haploview version 4.2. We used allele dose, dominant, and recessive genetic models to calculate association between genotypes and sepsis risk, using χ2 analysis. All P values were 2-sided, were adjusted using Bonferroni correction for multiple testing, with a P < 0.05 used to determine statistical significance.3. Ex vivo tumor necrosis factor α production.A human whole-blood assay was used as described by Majetschak et al [17]. In brief, whole blood mixed 1:1 with cell culture medium. Samples were prepared in duplicate. The mixtures were incubated at 37°C with endotoxin(lipopolysaccharide, 100 ng/m L) for 4 hours. After endotoxin stimulation, tumor necrosis factor-alpha(TNF-α) in the supernatants was assayed by a sandwich enzyme linked immunosorbent assay(ELISA). The detection limits of the assay were 4 pg/ml.ResultsConstruction of haplotype blocks and selection of tag SNPs.Eight SNPs(rs28362491, rs3774932, rs4648068, rs7119750, rs4803789, rs12609547, rs1560725 and rs842647) were identified as tag SNPs for the NF-κB family genes. All of them were shown to be high-frequent SNPs in this study cohort.Allele frequencies and genotype distribution of the 4 tag SNPs among trauma patients. The genotyping success rates of the four tag SNPs by pyrosequencing ranged from 98.3% to 100% in our study cohort. The MAFs among the 753 trauma patients were 41.8% demonstrated that the rs842647 polymorphism affect TNF-α production and might be used to estimate risk for sepsis and MODS in trauma patients. Further studies, both clinical and experimental, are therefore needed to confirm the significance of these findings and to investigate their synergistic effect with other genetic polymorphisms in relation to the development of sepsis in and the outcomes of trauma patients.(rs28362491), 44.7%(rs4648067), 37.6%(rs7119750) and 13.4%(rs842647), respectively, which were quite similar to those observed in the 137 unrelated CHB cohort in the Hap Map database. The genotype distribution of all four tag SNPs was in agreement with the Hardy-Weinberg equilibrium(P > 0.05)(Table 2), indicating that both allele and genotype frequencies of these tag SNPs in the population remain constant. That is to say, they are in equilibrium from generation to generation.Association of the 4 tag SNPs with sepsis risk and MODS score. After calculations using the Power and Sample Size software program, our sample(N = 753) was considered adequate to study the tag SNPs of the NF-κB family genes. There were no significant differences in age, sex ratio or ISS among patients stratified according to the different genotypes of each tag SNP. Among the four genetic variants selected in this study, only the rs842647, although it was found in only 15 patients with variant homozygotes in this study cohort, was shown to be significantly associated with the risk for development of sepsis and MODS in major trauma patients. We discovered a significant association between AA genotype of rs842647 and decreased susceptibility to sepsis(P = 0.024) and lower MOD scores(P = 0.013) in case of recessive model(AA vs. GA+GG). Data from multiple logistic regression analyses further indicated that the patients with the rs842647 polymorphism had a lower risk for developing sepsis(OR = 0.673, 95% CI = 0.532 to 0.873; P = 0.012) after adjusting for possible confounders, including age, sex ratio and ISS. There were no significant associations with sepsis morbidity rate and MOD scores for the other three tag SNPs(rs28362491, rs4648068 and rs7119750).Effect of rs842647 on LPS-induced TNF-α production. NF-κB is a key molecule for the maturation and secretion of TNF-α [18, 19]. Therefore, we hypothesized that the rs842647 polymorphism might be associated with TNF-α production. The rs842647 polymorphism was well associated with the LPS responsiveness of peripheral blood leukocytes. LPS-induced TNF-a production was significantly lower in patients with the variant A allele than that in those with wild G allele(P = 0.027 for recessive effect).ConclusionsIn the current study, we investigated the clinical relevance of the genetic variants within the entire NFKB1, RELA, and REL genes by means of tag SNPs. We have...