Functional Significance of Gene Polymorphisms in the Promoter of Myeloid Differentiation-2

Abstract

Objective:

To investigate the functional significance of the reported single nucleotide polymorphisms (SNPs) in the promoter of the myeloid differentiation-2 (MD-2) gene.

Summary Background Data:

Functional gene polymorphisms of innate immune receptors have been shown to be critical determinants of the immune inflammatory response. MD-2 is an important signaling enhancer protein in the endotoxin (LPS) receptor complex. Although a total of 156 SNPs have been identified within the whole MD-2 gene, little is known about the functional significance of these SNPs.

Methods:

The possible biosignificance of 8 reported SNPs was analyzed using on-line software tools. The selected SNPs were then genotyped using a restriction fragment length polymorphism method applied to 711 healthy Chinese volunteers. Their functional effects were assessed by the observation of transcription activity, MD-2 mRNA expression, and leukocyte response to ex vitro LPS stimulation. Moreover, the clinical relevance of these SNPs was investigated in 105 patients with major trauma.

Results:

Three SNPs (C–1625G, A-1064G, and A-475T) in the MD-2 promoter were selected based on bio-informatic analysis. Both −1625 and −1064 SNPs, rather than −475, were seen in the Chinese population, with frequencies of 19.8% (−1625G) and 34.7% (−1064G). But only the −1625 polymorphism was found to affect MD-2 promoter activity. Moreover, the expression of MD-2 mRNA and the production of TNF-α in whole blood leukocytes, in response to LPS stimulation, were significantly increased in subjects with the −1625 G allele. Patients who possessed the −1625 G allele were more likely to experience complications with organ dysfunction and sepsis after major trauma. All these associations were in allele-dose dependent effect.

Conclusions:

The MD-2/−1625 polymorphism is an important functional variant.

This study investigated the functional significance of the genetic variations in the promoter of the MD-2 gene and found that the −1625 polymorphism from the 8 reported polymorphic sites in the myeloid differentiation-2 promoter might be an important functional single nucleotide polymorphism.

Despite significant advances in both the development of therapeutic strategies and the understanding of its pathogenesis, sepsis remains a worldwide problem that is the leading cause of intensive care unit mortality.¹ Approximately 3 million people are diagnosed with sepsis in China each year, resulting in at least 600,000 deaths.² Although the pathophysiology of sepsis is complex, discovery of pattern recognition receptors (PRRs) on the surface of innate immune cells, and their interaction with pathogen-associated molecular patterns, have been important breakthroughs in understanding the molecular basis of this syndrome.^3–6 Lipopolysaccharide (LPS), a major component of the Gram-negative bacteria cell wall, is recognized in mammals by a receptor complex composed of CD14, Toll-like receptor (TLR)-4, and myeloid differentiation-2 (MD-2).^7,8 While CD14 is thought to bind LPS⁹ and TLR4 is thought to aid in LPS transmembrane signaling,¹⁰ MD-2 acts by enhancing the physical association of the extracellular region of TLR4 with LPS.^11,12 The essential role of MD-2 in LPS transmembrane signaling has been confirmed by studies demonstrating LPS hyporesponsiveness in cells that expressed TLR4 alone or TLR4 associated with mutant MD-2. Significantly, the decreased responsiveness could be rescued by transfection of MD-2 cDNA or soluble MD-2 protein.^13–15

Growing evidence indicates that variation within essential genes, including PRRs, cytokines, and coagulation factors, is an important determinant of immune inflammatory response to infection.^16–19 Our results, and those of others, have indicated that genetic polymorphisms within both the CD14 and TLR4 genes could significantly influence the expression of target genes, and are well associated with the susceptibility to, and outcome from, sepsis.^17,20,21 Human airway epithelial cells isolated from subjects with Asp299Gly and Thr399Ile polymorphisms in the coding region of the TLR4 gene are associated with a decreased response to inhaled endotoxin, which could be recovered via expressing wild-type TLR4 in these cells.²² The relevance of these TLR4 mutations was also shown in patients with gram-negative sepsis.^23–25 In addition, the CD14/−159 polymorphism is able to affect CD14 expression at both mRNA and protein levels,²⁶ and is clearly associated with occurrence of inflammatory disease.^27,28

Although 156 single nucleotide polymorphisms (SNPs) have been identified within the whole MD-2 gene as shown by public SNP database (dbSNP) (www.ncbi.nlm.nih.gov and www.ensembl.org), little is known about the functional significance of these SNPs. In view of the functional significance of genetic polymorphisms in CD14 and TLR4 genes, it is hypothesized that certain variants within the MD-2 gene may influence LPS-induced transmembrane signaling. The promoter, due to containing numerous transcription factor binding sites, is central to the regulation of gene transcription.²⁹ There is growing evidence indicating that genetic variations in this region might affect the transcription of target genes.^26,29,30 The promoter of MD-2 gene has been shown to be located within 2 kb-sequence upstream of the gene translational start site.³¹ Therefore, we selected the reported SNPs within the 3-kb sequence proximal to the MD-2 gene translational start site from the dbSNP in this study, and investigated their possible functional significance by observing their association with transcription factor binding sites, MD-2 mRNA expression, cellular activation, and outcome of patients with major trauma. In addition, this study also examined the effect of these polymorphisms on the promoter activity of the MD-2 gene.

MATERIALS AND METHODS

Study Population

A total of 816 unrelated adult Chinese, comprising 711 healthy volunteers and 105 patients with major trauma, were recruited in this study. All of them are Han Chinese and live in Chongqing, China. The 711 healthy volunteers, 533 men and 178 women, with a median age of 34 years (range, 18–55 years), were obligation blood donors. The 105 trauma patients (80 men and 25 women) were admitted to the department of trauma surgery in the Daping Hospital and the Chongqing Emergency Medical Center, Chongqing, China, between January 1, 2005 and June 1, 2006. They were enrolled in the study if they met the following criteria: 1) between 18 and 65 years of age, 2) expected Injury Severity Score (ISS) greater than 16, and 3) probability of survival greater than one week. Patients were not eligible if they had penetrating injuries or preexisting cardiovascular, respiratory, renal, hepatic, hematologic, or immunologic diseases. ISS was performed according to the abbreviated injury scale 1998 by independent evaluators.³² All patients requiring surgical intervention received standard surgical care and postoperative intensive care unit treatment. The protocol for this study was approved by the Ethical and Protocol Review Committee of the Third Military Medical University, and the informed consent was obtained from the subjects or the Patient’s next of kin.

Bioinformatic Analysis of SNPs

Eight SNPs in the 3-kb sequence proximal to the MD-2 gene translational start site were analyzed using online software tools to assess their association with repeat sequences (http://woody.embl-heidelberg.de/repeatmask) and to inspect them for any potential transcription factor binding sites (http://www.gene-regulation.com, and http://motif.genome.ad.jp).

Genotyping of SNPs

Genomic DNA was extracted from whole blood using the phenol/chloroform method, and analyzed using the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method. The primers for amplification were derived from published genomic sequences (Ensembl Gene ID: ENSG00000154589) (Table 1). The PCR reactions were run at 94°C for 5 minutes followed by 30 cycles of 30 seconds at 94°C, 40 seconds at 56°C, 40 seconds at 72°C. The reaction was completed by a final incubation at 72°C for 10 minutes. After digestion with specific restriction endonucleases (New England Biolabs, Beverly, MA), the PCR products were electrophoresed in a 2% agarose gel with subsequent staining by ethidium bromide. The bands were visualized under UV light and digitized with a video camera. The results were confirmed by 2 independent observers. In addition, RFLP results were further confirmed by DNA sequencing with 10 random samples (Takara Biotech), all of which agreed with genotypes obtained from RFLP analysis.

TABLE 1. Primers and Restriction Endonucleases for Genotyping of MD-2

Promoter Activity

The possible effect of both C−1625G and A–1064G SNPs on the promoter activity was investigated using a reporter gene assay system. A total of 4 plasmid constructs were prepared by inserting a 1765-bp sequence (−1704 ∼ +61) of the MD-2 gene into a promoterless pGL3-Basic vector (Promega, Madison, WI), which contained a combined wild genotype of −1625C and −1064A, or a combined mutant genotype of −1625G and −1064G, or a single mutant genotype of either −1625G or −1064G. The 4 constructed vectors (2 μg DNA) were transiently transfected into Human U937 cells using FuGENE6 reagent (Roche Molecular Biochemicals, Indianapolis, IN). At 24 hours post-transfection, the cells were treated with LPS (100 ng/mL) for 24 hours. Luciferase activities were measured with a Luminoskan Ascent luminometer (Thermo Labsystems, Helsinki, Finland). Luminescence experiments were performed at least 3 times, with each transfection in triplicate, using 6 separate DNA preparations. Results are expressed as fold increase in relative luciferase activity (RLA) of the MD-2 promoter construct vectors compared with the RLA of pGL3-Basic.

Ex Vivo LPS Stimulation of Whole Blood

A human whole-blood assay was used as described by Majetschak et al.³³ In brief, aliquots of whole blood collected from the healthy volunteers were mixed 1:1 with RPMI 1640 culture medium, and incubated with 100 ng/mL LPS (Escherichia coli O26:B6, Difco Laboratories, Detroit, MI) in a sample mixer at 37°C for 4 hours.

MD-2 mRNA Expression

After LPS incubation, the total RNA was isolated from whole blood leukocytes using Revertaid (MBI Fermentas, Vilnius, Lithuania). Expression of MD-2 mRNA was quantified by real-time quantitative PCR with the TaqMan gene expression assay (Takara Biotech).³⁴ Primes were 5′ TTC CAC CCT GTT TTC TTC CAT3′, 5′ TTG GGT ATT GCA TTT TAT CAC AG3′ for MD-2, and 5′ ACA GCC TCA AGA TCA TCA GCA 3′, 5′ ATG AGT CCT TCC ACG ATA CCA 3′ for the internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Probes for both MD-2 and GAPDH were 5′FAM- TGA AGC TCA GAA GCA GTA TTG GGT CT –TAMRA3′ and 5′FAM– GTG CTA AGC AGT TGG TGG TGC AGG A –TAMRA 3′, respectively. Every mRNA target was amplified in duplicate in separate wells. After reverse transcription for 1 hour at 42°C, and an inactivation step for reverse transcription of 10 minutes at 95°C, 45 cycles of 5 minutes at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C were performed using QPCR (iCycler Optical Module 584BR, BIO-RAD). Relative expression intensity of MD-2 mRNA was normalized as the ratio of MD-2 to GAPDH and calculated with the formula of 2 power (−ΔΔC_t). Data are expressed as fold increase (2^−ΔΔCt) compared with the value of CC genotype.

Tumor Necrosis Factor-α Production

The supernatants were collected by centrifugation after above ex vivo LPS stimulation and stored at −70°C until use. Tumor necrosis factor-α (TNF-α) levels were assayed by a sandwich enzyme linked immunosorbent assay, according to the manufacturer’s instructions (Endogen, Woburn, MA).

Clinical Evaluation

After admission, the patients with major trauma were monitored in the following 6 aspects: respiratory (PO₂ /FIO₂ ratio), renal (serum creatinine concentration), hepatic(serum bilirubin concentration), cardiovascular (pressure-adjusted heart rate), hematologic (platelet count), and central nervous (Glasgow Coma Scale) systems. The organ function was then scored using the method of Marshall et al.³⁵ Sepsis was defined if patients met all the following criteria: clinical evidence of infection, body temperature greater than 38.5°C or less than 36.5°C, and leukocyte count greater than 10 × 10⁹/L or less than 4 × 10⁹/L.

Statistical Analysis

Allele frequencies for each SNP were determined by gene counting. Genotype distribution was tested for departure from Hardy-Weinberg equilibrium using χ² analysis. The extent of pairwise linkage disequilibrium was determined using the random-permutation procedure implemented in the Arlequin package. Luciferase expression was compared using independent-sample t test. A one-sample Wilcoxon signed-rank test was performed for MD-2 mRNA expression by comparing the median values to the hypothetical median of 1 (1-fold increase). Difference in plasma TNF-α levels was evaluated by means of ANOVA. The association between the −1625 polymorphism 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, such as allele dose model, dominant model, or recessive model, were used. For dominant effect, we compared the G allele carriers (CG and GG genotypes) versus noncarriers (CC genotype); whereas for recessive effect, subjects homozygous for the G allele were compared with heterozygous (CG) carriers and noncarriers (CC). Allele dose was defined as the number of copies of minor allele (G) in the genotype. We performed linear regression analysis to quantify the allele dose effect. The association of genotypes with sepsis morbidity rate was determined by χ² analysis. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated by multiple logistical regression analyses to estimate the relative risk of sepsis. Results were considered to be significant if P < 0.05. All statistic analysis was carried out using SPSS Version 11.0 (Chicago, IL).

RESULTS

Bioinformatic Analysis of SNPs in the MD-2 Promoter

The DNA sequence from position MD-2/−3000 to MD-2/+1, covering the entire 8 polymorphic sites (G-2448A, G-2190A, A-2059T, C−1625G, C-1201G, G-1174T, A-1064G, and A-475T), was inspected for any relationship of their location with repeat sequences and binding sites of potential transcription factors using online software tools. Table 2 shows that 4 of the 8 polymorphic sites (−2448, −2059, −1201, and −1174) were located in repeat sequences, suggesting that these polymorphisms may be of less functional significance. Among the rest 4 sites, 3 sites (−1625, −1604, and −475) were shown to be located on transcription factor binding sites, such as AP4, MyoD for −1625, NKx-2.5, SREBP-1, Cap, delta EF1, SREBP-1, Lmo2 for −1064, and CdxA, OCT1 for −475 locus. Another site (−2190) was not related to any transcriptional factor binding site. Therefore, we selected the −1625, −1604, and −475 polymorphic sites from the 8 variants in the MD-2 promoter for further study.

TABLE 2. Bioinformatic Analysis of 8 Polymorphic Sites in the MD-2 Promoter

Allele Frequencies and Genotype Distribution

We first observed the occurrence of the above selected 3 polymorphic sites in Chinese Han population (n = 710). Two of them were identified in our study group. They were C−1625G and A-1064G. No genetic variation was observed at position −475. The allele frequencies were 80.2% and 19.8% for the C and G alleles at position −1625, and 65.1% and 34.9% for the A and G alleles at position −1064, respectively. The genotype distribution for both SNPs was found to be in Hardy-Weinberg equilibrium (P = 0.963 for −1625 and P = 0.955 for −1064). The 2 polymorphic sites were in tight linkage disequilibrium (D′ = 0.7, r² = 0.56, P < 0.0001).

Effect of the MD-2 Promoter Polymorphisms on Transcription Activity

The possible functional significance of the MD-2/−1625 and −1064 SNPs were then examined by observing their effect on the promoter activity of the MD-2 gene. Figure 1 shows that the C→G variation at position −1625 could significantly enhance luciferase activities comparing to the wild haplotype (−1625C/−1064A) (1.95 ± 0.14 vs. 1.66 ± 0.07, P = 0.01). But the A→G variation at position −1064 appears to have no marked effect (1.71 ± 0.09 vs. 1.66 ± 0.07, P = 0.39). The luciferase activities were not significantly further increased in case of coexistence of the 2-point variants (−1625G/−1064G haplotype) when compared with only the −1625 variant (1.98 ± 0.14 vs. 1.95 ± 0.14, P = 0.746). The results indicated that only the genetic variation at position −1625 could affect the promoter activity of the MD-2 gene.

FIGURE 1. Effect of the MD-2/−1625 and −1064 polymorphisms on transcription activity. Relative luciferase activity (RLA) was assayed in cells transfected with −1625C/−1064A, −1625G/−1064A, −1625C/−1064G or −1625G/−1064G plasmid construct. Luciferase activity was normalized for transfection efficiency using a control plasmid pRL-CMV. Results are expressed as fold increase in RLA of the MD-2 promoter construct vector as compared with pGL3-Basic. Luciferase expression was compared using independent-sample t test *P < 0.05 compared with −1625C/−1064A haplotype.

Effect of the MD-2/−1625 Polymorphism on LPS-Induced Expression of MD-2 mRNA

In view of transcriptional activity of the MD-2/−1625 SNP, we further hypothesized that this genetic variant would influence target gene expression and LPS-induced cell activation. We selected 36 subjects from the 711 healthy volunteers (n = 12 for each genotype). There was no significant difference in spontaneous expression of MD-2 mRNA in peripheral blood leukocytes among different genotypes. But inducible expression was shown to be significantly different when stimulated with LPS (Fig. 2). This association appeared to be dose dependent, with 1.75-fold increase in CG and 2.81-fold increase in GG genotypes. A significant difference exists between the CC and GG genotypes (P < 0.05).

FIGURE 2. Effect of the MD-2/−1625 polymorphism on LPS-induced MD-2 mRNA expression. The whole blood samples from 36 healthy volunteers (n = 12 for each genotype) were mixed 1:1 (vol/vol) with RPMI 1640 culture medium and incubated with 100 ng/mL of Escherichia coli LPS (O26:B6) at 37°C. After 4 hours of incubation, the total RNA was isolated from whole blood leukocytes and MD-2 mRNA expression was determined by quantitative real-time PCR. Data are expressed as fold increase (2^−ΔΔCt) compared with the value of CC genotype. The reference gene was glyceraldehyde-3-phosphate dehydrogenase. Values are mean ± SD of 12 subjects. A one-sample Wilcoxon signed-rank test was performed. *P < 0.05 versus the CC genotype.

Effect of the MD-2/−1625 Polymorphism on LPS-Induced TNF-α Production

Figure 3 further shows that the MD-2/−1625 polymorphism was well associated with the activation of blood leukocytes by ex vivo LPS stimulation. Although baseline levels of plasma TNF-α were not significantly different among different genotypes, LPS-induced TNF-α production was significantly increased in subjects with CG genotype (887 ± 640 pg/mL) and GG genotype (1438 ± 784 pg/mL) comparing to those in subjects with CC genotype (437 ± 258 pg/mL) (P < 0.001 for both dominant and recessive effects). In addition, this association with inducible TNF-α production was significantly allele-dose dependent as indicated by linear regression analysis (P < 0.001).

FIGURE 3. Effect of the MD-2/−1625 polymorphism on LPS-induced TNF-α production. The whole blood samples from 75 healthy volunteers (n = 25 for each genotype) were treated as the same to the above. TNF-α levels in the supernatants were assayed by a sandwich enzyme linked immunosorbent assay. One-way ANOVA was used to assess statistical significance. a = P < 0.001 for dominant association (GG + CG vs. CC). b = P < 0.001 for recessive association (GG vs. CG + CC).

Association of the MD-2/−1625 Polymorphism With Clinical Phenotypes of Patients With Major Trauma

In view of the functional effect of the −1625 SNP shown by the above results, we further investigated the clinical relevance of this SNP in 105 patients with major trauma (ISS, 22.6 ± 7.8). There were no significant differences in age, gender ratio, or ISS scores among patients stratified according to the different genotypes of the −1625 locus (P > 0.05, Table 3). The overall allele frequencies and genotype distribution of this polymorphism in trauma patients were similar to those in healthy volunteers, showing 77.6% for the C allele and 22.4% for the G allele, respectively. MOD scores in trauma patients were well associated with the −1625 polymorphism, showing a significant difference in case of dominant effect (P < 0.001), and a borderline significant difference in case of recessive effect (P = 0.092). Our data from linear regression analysis indicated that this association with MOD scores was significantly allele-dose dependent (P = 0.001). Moreover, trauma patients carrying the G allele appeared to have higher risk of sepsis comparing to those carrying the C allele (OR, 6.49; 95% CI, 1.25–33.72, P = 0.026). There was significant difference in sepsis morbidity in case of dominant effect (P = 0.022), and a borderline significant difference in case of recessive effect (P = 0.087). The association of this polymorphism with MOD scores and sepsis morbidity was not influenced by age, gender ratio, and ISS.

TABLE 3. Characteristics of Patients With Severe Trauma

DISCUSSION

To the best of our knowledge, the present study provides the first evidence of the functional significance of gene polymorphisms in the MD-2 promoter. Three polymorphisms (−1625C→G, −1064A→G and −475A→T) were selected from a total of 8 SNPs in the promoter of MD-2 gene. Each of the 3 SNPs chosen for further study, as shown by bioinformatics analysis, locates in nonrepeat sequences and has a close relationship with binding sites of important transcription factors. It suggests that the 3 SNPs might have a functional significance.

In view of the potential functional significance of the −1625, −1064, and −475 SNPs, we then investigated their occurrence in Chinese Han population. A total of 710 healthy volunteers were recruited in this study. To avoid an artifact in population admixture,³⁶ all of them are Han Chinese who live in Chongqing district. The genotype frequency distribution is shown to be in Hardy-Weinberg equilibrium, further suggesting that our study population is homogeneous. Our results indicate that both MD-2/−1625 and −1064 variants exist in the Chinese Han population with a high frequency, showing 19.8% and 34.7% for the −1625 G and −1064 G alleles, respectively. This is quite similar to the reported frequencies in dbSNP (Table 2). We did not identify the genetic variation at position −475 in the study group. The allele frequency for this polymorphic site is not shown in dbSNP. Our observations, together with dbSNP report, indicate that the MD-2/−1625 and −1064 polymorphisms, rather than the −475, are common variants that exist in both Chinese Han and Western populations.

In vitro transfection cell model with plasmid constructs containing tested alleles is a powerful approach to elucidate the functional effects of gene polymorphism.³⁷ Using this model, we then examined the direct effect of the MD-2/−1625 and −1064 polymorphisms on the MD-2 promoter activity. It is found that the C→G variation at position −1625 could significantly enhance the transcription activity of the MD-2 promoter, whereas the A→G variation at position −1064 has little effect. Although both SNPs are shown to be in tight linkage disequilibrium, there appears no marked interaction between these 2 SNPs, showing no significant difference in fold increase in RLA between cells transfected with a single mutation at position −1625 (−1625G) and combined mutation at positions −1625 and −1064 (−1625G/−1064G). The results suggest that the MD-2/−1625 polymorphism may be a causal variant, while the MD-2/−1064 polymorphism may be of less functional significance.

Given the effect of the −1625 polymorphism on transcription activity, we hypothesized that this genetic variation might influence the expression of the MD-2 gene. Our results indicate that although there is no significant difference in spontaneous expression of MD-2 mRNA among subjects with different genotypes, there is an increasing trend for inducible expression of MD-2 mRNA in association with G allele copy number. The inducible MD-2 mRNA expression is shown to be significantly increased in subjects with the GG genotype when compared with subjects with the CC genotype. It suggests that the C→G variation at position −1625 not only affect the transcriptional activity of the MD-2 promoter but also enhance the expression of the MD-2 gene.

MD-2 has been recognized as one of pivotal PRRs that elicit coordinated innate immune defenses through recognition of microbial products and induction of inflammatory mediators.^10–14 The levels of MD-2 expression are thought to be an important determinant for LPS-induced production of proinflammatory cytokines.^38–40 Based on our results, we hypothesized that the −1625 SNP-mediated change of MD-2 expression might influence LPS-induced activation of innate immune cells. TNF-α is a cytokine commonly used to assess LPS-induced cellular activation both in vivo and in vitro. Our results show that, although there is no significant difference in spontaneous TNF-α production among subjects with different genotypes, TNF-α production in response to ex vivo LPS stimulation is significantly different, showing that plasma TNF-α levels are significantly elevated in subjects with the −1625 G allele than in those with the −1625 C allele. Linear regression analysis further indicates that this association is allele-dose dependent. These results further demonstrate that the genetic variation at position −1625 in the MD-2 promoter is an important functional SNP, not only increasing the transcription activity of the MD-2 promoter and the expression of MD-2 gene, but also enhancing LPS-induced activation of innate immune cells. MD-2 expression has been recognized to be a useful marker to predict the clinical outcome of patients with sepsis.^41–43 We therefore reason that the MD-2/−1625 polymorphism might be used as genetic marker to predict susceptibility to sepsis in critically ill patients.

Trauma is a major public health problem worldwide, ranking as the fourth leading cause of death among all diseases.⁴⁴ One of the most serious complications occurring as a consequence of major trauma is the sequential dysfunction of multiple organs, which in the majority of cases is associated with post-traumatic sepsis.⁴⁵ The intestine has been considered as a major source of bacteria and lipopolysaccharide for causing sepsis in major trauma patients.^46,47 The expression of PRRs, such as CD14, TLR2, TLR4, and MD-2, have been shown to be elevated after injury or hemorrhagic shock.^42,48–50 This is considered as an important mechanism for that injury primes the innate immune system,^48,51,52 leading to overwhelming proinflammatory response when bacteria and their products enter into body, and then causing sepsis and multiple organ dysfunction.^53–55 In view of the functional effect of the MD-2/−1625 polymorphism observed in this study, we hypothesized that this polymorphism might be associated with predisposition to vital organ dysfunction and sepsis in patients with major trauma. A total of 105 patients with major trauma were stratified according to the different genotypes of the −1625 polymorphism. Age, sex, and injury severity of trauma patients did not differ statistically between different genotypes. The overall allele frequencies and genotype distribution of the −1625 polymorphism in trauma patients were consistent with those in the 710 healthy volunteers, suggesting that there may not exist any artifact due to population heterogeneity in our studied group. Our results indicate that trauma patients with the G allele have higher risk of sepsis comparing to those carrying the C allele (OR, 6.49; 95% CI, 1.25–33.72, P = 0.026). There is significant difference in sepsis morbidity among patients with different genotypes. MD-2 is an essential part of receptor complex for LPS recognition and plays an important role in antimicrobial defense by mediating the production of antimicrobial peptides.^56,57 MD-2 is also essential for initiating and aggravating LPS-induced inflammatory response.^11,12 To this end, blockade of MD-2 could protect mice from sepsis.⁵⁸ Mice lacking MD-2 do not respond to LPS and are resistant to endotoxin shock.^11,41 These suggest that the MD-2/−1625 polymorphism may increase susceptibility to sepsis through enhancement of MD-2 expression and LPS-induced inflammatory response. Similarly, MOD scores in trauma patients with the −1625 G allele are significantly higher than those in patients with the C allele. This association possesses a significant allele-dose effect and does not change after adjustment for potential confounders, such as age, gender ratio, and injury severity. In combination with the central role of MD-2 in the pathogenesis of sepsis, the MD-2/−1625 polymorphism may be considered as an important genetic risk factor for susceptibility to post-traumatic sepsis and vital organ dysfunction. Future studies in larger population and on assaying plasma inflammatory cytokines in trauma patients are needed to confirm the relevance of the C−1625G polymorphism.

ACKNOWLEDGMENTS

The authors thank Dr. Hong Dong, Ce Yang, and Ms. Qin Liu for their technical help and writing assistance, and Dr. Qi Wan and Dr. John Mielke, University of Toronto, for reviewing the article.