Identification Of Tag Single Nucleotide Polymorphisms Within Some TLR Genes And Their Clinical Relevance

Background:The host response to infection is initiated by the early response of the innate immune system to an array of conserved pathogen-associated molecular patterns. Toll like receptors (TLRs) are evolutionarily conserved pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns. It has been demonstrated that TLRs confer specificity to the innate immune response through their extracellular domain to recognize different pathogen-associated molecular patterns (PAMPs) of microbes, such as Toll-like receptor 2 (TLR2) responsible for bacterial lipoprotein, TLR4 for the recognition of bacterial lipopolysaccharide, TLR9 for bacterial DNA. Clinical observations have revealed that the susceptibility to microbial pathogens varies between individuals. Unequivocal evidence shows that genetic factors may be important determinants for the inter-individual differences in susceptibility to infection. Our previous results have indicated that genetic polymorphisms within some PRR genes, such as CD14, myeloid differentiation-2 (MD-2) and could be well associated with the susceptibility to, and outcome from, sepsis in severe trauma patients. Increasing number of studies suggest that genetic variation in the TLR2, TLR4 and TLR9 genes may be associated with susceptibility to infectious diseases. Therefore, we speculated some genetic polymorphisms of the TLR2, TLR4 and TLR9 genes might be considered as relevant risk estimate for organ dysfunction and sepsis in trauma patients. To our knowledge, little is known about the clinical relevance of TLR2, TLR4 and TLR9 polymorphisms in trauma patients.Materials and Methods:1. Study population. A total of 557 unrelated patients with major trauma (Injury Severity Score more than 16) were recruited in this study. The patients consisted of 331 men and 79 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 5, 2009. Their average age was 38.4 and median ISS was 25.8. 2. Bioinformatics analysis. Genetic variation data for the entire TLR2 and TLR9 genes was obtained from the HapMap project for 45 Chinese Han Beijing (CHB) population (www.hapmap.org). Haplotype blocks were constructed throughout the whole TLR2 and TLR9 gene using Haploview. A maximally informative haplotype tag SNP (htSNP) was then selected from each block using software Tagger program.3. Genotyping of htSNPs. SNPs were detected by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and pyrosequencing methods.4. Assay of cytokine production. The whole blood collected from the severe trauma patients within 24 hrs after admission. The cytokines'levels in the supernatants were determined with enzyme-linked immunosorbent assay according to the manufacturer's instructions.5. Clinical evaluation. Sepsis was defined if patients met all the following criteria: clinical evidence of infection, body temperature greater than 38.5℃or less than 36.5℃, and leukocyte count greater than 10×109/L or less than 4×109/L. The organ function was scored using the method of Marshall JC and calculated as a single daily value during the ICU stay. The MOD scores and sepsis were determined by individuals who did not know the genotypes.6. Statistical Analysis. Genotype distribution of each SNP was tested for departure from Hardy-Weinberg equilibrium (HWE) usingχ2 analyses. The extent of pair-wise linkage disequilibrium between polymorphisms was determined by the Haploview software. The association between polymorphisms and cytokine levels was determined using one-way analysis of variance. The association between polymorphisms and MOD scores was performed using analysis of covariance testing with age, gender ratio, and injury severity to adjust for possible confounding effects. Three genetic models (allele-dose, dominant, and recessive) were used. The association of genotypes with sepsis morbidity rate was determined byχ2 analysis. Odds ratios with 95% confidence intervals were calculated by multiple logistic regression analyses to estimate the relative risk of sepsis.Results:1. Bioinformatics analysis. Two haplotype blocks were constructed within the full TLR2 gene in Chinese Han Beijing population. Subsequently, a total of three htSNPs (3013 A/G, 19216 T/C and 22215 G/T) were selected in the TLR2 gene. One haplotype block was constructed within the TLR9 gene. TLR9 -1486 A/G were selected as htSNP.2. Allele frequencies and genotype distribution of the SNPs among trauma patients. The minor allele frequencies of the three htSNPs in the TLR2 gene among 410 trauma patients in this cohort were 39.5%, 26.5% and 47.4%, respectively. The MAF of the TLR4/-2242 polymorphism in 303 trauma patients was 42.9%. The MAFs of the four key SNPs(-1486 A/G, 1174 A/G, 2849 C/T and 6577 T/C) in the TLR9 gene among 557 trauma patients were 38.8%, 36.9%, 37.7% and 36.3%. However, TLR9 -1237 T/C was removed in this study due to its too low frequency (0.8%). The genotype distributions of the SNPs in our study were all in agreement with the Hardy-Weinberg equilibrium.3. Association of the SNPs with the capacity of peripheral leukocytes to produce cytokines. TLR2 3013 A/G, 19216 T/C SNPs and the number of ATT haplotype copy were shown to be well associated with the capacity of peripheral leukocytes to produce TNFα, IL-8 and IL-10 in response to lipoprotein stimulation. Results revealed that TLR9 -1486 A/G, 2849 C/T, 6577 T/C and the number of GGTC haplotype copy were well associated with the TNFαlevel in peripheral blood in 24h after trauma.4. Association of the SNPs with development of sepsis and MODS in patients with major trauma. For the TLR2 19216 T/C, TLR4 -2242 T/C and TLR9 6577 T/C, there were significant differences in sepsis morbidity rate and MOD scores among the patients with different genotypes. The number of ATT and GGTC haplotype copy were well associated with sepsis morbidity rate and MOD scores, respectively.Conclusion:1. In Chinese Han Beijing population, two haplotype blocks were constructed within the full TLR2 gene. And three htSNPs (3013 A/G, 19216 T/C and 22215 G/T) were identified in the TLR2 gene. There was only one haplotype block within the TLR9 gene. TLR9 -1486 A/G were selected as htSNP.2. The three htSNPs (3013 A/G, 19216 T/C and 22215 G/T) in the TLR2 gene, TLR4 -2242 T/C and the four key SNPs(-1486 A/G, 1174 A/G, 2849 C/T and 6577 T/C) in the TLR9 gene do exist in Chongqing Han population, with high frequency. TLR9 -1237 T/C SNP do not exist in this cohort.3. The htSNPs 3013 A, 19216 T, or the wild haplotype ATT might attenuate TLR2-mediated activation of innate immune cells. The genetic variation at positions TLR9 -1486 G, 2849 T, 6577 C and haplotype GGTC might enhance the mediation of TLR9 on immune cells after severe trauma.4. TLR2 19216 C, TLR4 -2242 C and TLR9 6577 C might be used as risk determinants for sepsis and MODS in trauma patients. The haplotype ATT in TLR2 gene and haplotype GGTC in TLR9 gene might be used as genetic biomarkers for the assessment of susceptibility to inflammatory diseases, such as sepsis and multiple organ dysfunctions secondary to severe trauma..