Sepsis after trauma has a complex pathophysiology that involves both the pathogenic microbe and the patient's individualized immune response. Several single nucleotide polymorphisms (SNPs) are known to influence a patient's susceptibility to and recovery from sepsis. Many of these SNPs are in inflammation-related genes (1), such as lipopolysaccharide binding protein (2), interleukin-1 (3), CD14 (4), toll-like receptors (5,6), and tumor necrosis factor-beta (TNF-β) (7). By understanding genetic variation that influences response to injury, we will move closer to personalized or “precision medicine”—improving our ability to deliver the right treatment to the right patient at the right time.
The melanocortin-1 receptor (MC1R) is an anti-inflammatory mediator (8). It is a G protein-coupled receptor that binds with equal affinity to α-melanocyte-stimulating hormone (α-MSH) and adrenocorticotropic hormone (8). The receptor is expressed on numerous cells involved in the inflammatory response, including neutrophils, leukocytes, macrophages, endothelial cells, and melanocytes. Activation of the receptor results in a wide range of anti-inflammatory effects, including attenuation of macrophage cytokine production (9), reduction of leukocyte adhesion and migration (10), and inhibition of neutrophil chemotaxis (11).
The MC1R gene is highly polymorphic, with many functional variants (12). Several MC1R SNPs are associated with diseases such as melanoma (13), nonmelanoma skin cancers (14), and multiple sclerosis (15). We previously reported that the MC1RR163Q variant was associated with a higher risk of hypertrophic scarring after burn injury (16). Given the immunologic role of MC1R, we hypothesized that its common functional SNPs (Fig. 1) would be associated with the risk of developing complicated sepsis after trauma.
PATIENTS AND METHODS
Study design, population, and setting
We performed a retrospective cohort study using samples and data from a prior Institutional Review Board (IRB)-approved prospective study that enrolled subjects admitted to the trauma intensive care unit (ICU) at a regional Level 1 trauma center, between 2003 and 2005, as previously described (5,17). Patients were excluded if they were in the ICU less than 48 h, had an isolated traumatic brain injury, or were expected to die from their injuries. Injury severity score (ISS) was obtained from a prospectively acquired trauma registry. Admission and clinical data were acquired from electronic medical records with IRB approval.
Exposures and outcomes
The exposures of interest were one of eight different MC1R SNP genotypes. Assessed outcomes included lower respiratory tract infection, defined as a quantitative protected bronchial culture demonstrating at least 105 colony forming units/mL; bacteremia, defined as bacterial growth in a blood culture; urinary tract infection, defined as a urine culture with greater than 105 organisms per high power field; wound infections, defined by clinical diagnosis with culture confirmation when available; and complicated sepsis, defined as severe sepsis or septic shock according to criteria established by the American College of Chest Physicians/Society of Critical Care Medicine Consensus Committee (18).
DNA was isolated from discarded venous blood with QIAamp DNA Blood Midi Kits (Qiagen, Valencia, Calif). Genotyping was performed on 20 ng DNA samples using predesigned TaqMan SNP Genotyping Assays (Qiagen) in 384-well plates with a Viia 7 Real-Time PCR System (Applied Biosystems, Foster City, Calif) per manufacturer guidelines. The samples were genotyped for the following MC1R SNPs: MC1RV60L (rs1805005), MC1RD84E (rs1805006), MC1RV92M (rs2228479), MC1RR142H (rs11547464), MC1RR151C (rs1805007), MC1RR163Q (rs885479), MC1RD294H (rs1805009), and MC1RT314T (rs2228478) (Fig. 1). SNPs with a minor allele frequency (MAF) 1% or less were excluded from the analysis.
Genome-wide genotyping and principal components analysis
A subgroup of 511 Caucasian subjects had genome-wide genotyping data available from a previous study that investigated the genetic risks for ventilator-associated pneumonia (Wurfel and O’Keefe, manuscript in preparation). These subjects had required mechanical ventilation for at least 2 days. We did not test the SNPs from this genome-wide data for associations with complicated sepsis; instead, we used these SNPs as “null markers” of population substructure (19). The “null markers” were transformed into principal components, and the principal components were included as covariates in the regression analyses.
We determined the genotypes for approximately 620,901 polymorphisms. Each chip was stained and imaged on an Illumina Bead Array Reader. Normalized intensity data for each sample were loaded into Illumina Beadstudio 2.0 and genotypes called using the manufacturer's clustering algorithm. Dataset quality control was performed using SNP and Variation Suite 7.2 (Golden Helix, Bozeman, Mont) and PLINK (20). We followed a published protocol for quality control in genetic case-control association studies (21). Samples with sex discordance as determined by X heterozygosity, excess relatedness, and those with an autosomal heterozygosity rate ±3 SDs from the mean were excluded. Genetic markers were then filtered for call rate less than 0.95, MAF less than 0.05, and for deviations from Hardy-Weinberg Equilibrium (HWE) in the control population (P < 0.005). A total of 480,811 SNPs passed quality control filtering. We then performed principal components analysis to determine eigenvalues.
Associations between the MC1R SNP genotypes and outcomes were evaluated with univariate and multivariate logistic regression. We assumed any contribution to the sepsis phenotype from individual MC1R SNPs would be additive. Therefore, genotypes were coded as 0 (common allele homozygous), 1 (heterozygous), or 2 (variant allele homozygous). A univariate and multivariate model was fit for each SNP individually. Multivariate models included the following covariates: age and body mass index (BMI), modeled as continuous variables; sex, ISS (ISS <15 or ISS ≥15), and packed red blood cell (PRBC) transfusion requirement (<10 or ≥10 units PRBC transfusion during hospitalization) modeled as binary variables; and mechanism of injury (blunt, penetrating, or burn) modeled as a categorical variable. HWE was determined by chi-square test. In the subgroup of subjects with genome-wide data, eigenvalues for the first 10 principal components (see above) were included in the model to adjust for population substructure.
To account for multiple testing, all P value thresholds for significance were adjusted by the Bonferroni correction to control the familywise error rate at 0.05. Logistic regression analysis was performed using Stata 11.2 statistical software (StataCorp LP, College Station, Tex). Haplotype estimation and linkage disequilibrium analyses were performed using the Haplo Stats and LDheatmap packages, respectively, in R version 3.0.2.
Demographics and clinical outcomes
Figure 2 diagrams study enrollment and the cohorts that were analyzed. In total, 1,961 subjects were enrolled. To minimize confounding by population substructure, only Caucasian subjects with complete clinical and MC1R genotyping data were included in the analysis (n = 1,246). The demographics and clinical outcomes of this cohort are shown in Table 1. The majority were male (70%), severely injured (81%), and injured by a blunt mechanism (89%). Four hundred ninety-seven (40%) developed sepsis and 290 (23%) developed complicated sepsis. Overall mortality was 9%, and this was higher among subjects with sepsis (11%) and complicated sepsis (14%).
Characterization of variation and haplotype structure of the MCR1 gene
The technical genotyping success rate was at least 99% for all eight MC1R SNP genotyping assays. Genotype frequencies and MAFs among Caucasians with complete clinical data (n = 1,246) are shown in Table 2. MC1RV60L was the most common SNP with an MAF of 12%. MC1RR163Q had an MAF of 6%. MC1RD84E and MC1RR142H had MAFs of 1% or less, and were therefore excluded from further analysis, leaving six SNPs (MC1RV60L, MC1RV92M, MC1RR151C, MC1RR163Q, MC1RD294H, and MC1RT314T) for association testing. In haplotype analysis only MC1RV92M and MC1RT314T had a relevant degree of linkage disequilibrium (R2 = 0.74) (Fig. 3), with a MC1RV92M/MC1RT314T haplotype frequency of 8.4%.
The MC1RR163Q variant was associated with a lower risk of complicated sepsis
The incidence of complicated sepsis varied by genotype, ranging from 9% among MC1R163QQ homozygotes to 50% among MC1R151CC homozygotes. Unadjusted and adjusted analyses of associations between the MCR1 SNPs and complicated sepsis are shown in Table 3. Six SNPs were analyzed, so the Bonferroni-adjusted P value threshold for significance was 0.05/6 = 0.008. In univariate analysis, the MC1RR163Q variant was associated with a significantly lower risk of complicated sepsis (odds ratio [OR] = 0.42, 95% confidence interval [CI]: 0.26–0.69, P = 0.001). This association persisted after adjusting for age, sex, ISS, PRBC transfusion requirement, BMI, and mechanism of injury (adjusted odds ratio [ORadj] = 0.48, 95% CI: 0.28–0.81, P = 0.006) (Table 3). There were no associations between the other MC1R SNPs and complicated sepsis in univariate or multivariate analyses. In addition, there were no associations between complicated sepsis and MC1R haplotypes that did not include the MC1RR163Q variant. Notably, the MC1RR163Q variant was not associated with the risk of developing an infection (ORadj = 0.78, 95% CI: 0.56–1.09, P = 0.142) or sepsis without organ dysfunction (ORadj = 0.77, 95% CI: 0.54–1.11, P = 0.158).
The MC1RR163Q variant was associated with a lower risk of complicated sepsis after adjusting for population substructure
All SNPs were in HWE (P > 0.05), with the exceptions of MC1RR163Q (P = 0.005) and MC1RD294H (P < 0.001). The departure from HWE raised the possibility that the association between MC1RR163Q was an artifact of population substructure. We used principal components to adjust for this potential substructure in a subset of subjects (n = 511) who had genome-wide data from a previous study (Wurfel and O’Keefe, manuscript in preparation). The characteristics of this subgroup are detailed in Supplemental Table 1, http://links.lww.com/SHK/A434. In regression analysis, principal components did not attenuate the association between the MC1RR163Q variant and lower risk of complicated sepsis (ORadj = 0.30, 95% CI: 0.13–0.70, P = 0.005).
Our results suggest that variation in the MC1R gene is associated with the severity of sepsis after traumatic injury. Subjects with the MC1RR163Q variant had a lower risk of developing complicated sepsis. The variant did not seem to influence the risk of developing an infection, but rather seemed to influence the host response to a nosocomial infection. This is consistent with other studies of the relationship between genetic variation and infection severity (22).
The mechanism for the inverse association between the MC1RR163Q variant and complicated sepsis is unclear, but this SNP may be a causal factor behind the improved outcomes. Several previous studies showed that the melanocortin system mediates inflammation and plays a role in sepsis. In an in vivo study, mice with defective MC1R signaling had more severe vascular dysfunction in response to lipopolysaccharide (LPS) than did wild-type mice (23). In another study, mice treated with α-MSH had less liver damage in response to LPS than untreated mice, an effect that was mediated by decreased leukocyte infiltration and cytokine accumulation (24). In mice with sepsis induced by cecal ligation and perforation, an α-MSH analogue decreased hemodynamic failure, acute kidney injury, and mortality (25). Moreover, α-MSH has been associated with the inflammatory response and critical illness in humans (26). In healthy subjects, endotoxin administration resulted in increased serum α-MSH concentrations (27). In critically ill subjects with sepsis, higher serum α-MSH concentrations were associated with lower serum TNF-α concentrations, and septic patients with persistently low α-MSH concentrations had a higher risk of death (26). Taken together, these studies indicated that the melanocortin system participates in the innate immune response and that experimental genetic variation alters responses. Therefore, it is possible that the association we observed between an MC1R variant and complicated sepsis is due to a functional change in the receptor.
MC1R signaling involves two pathways: a cyclic AMP (cAMP) pathway that is activated by adenylate cyclases (28), and a cAMP-independent pathway involving mitogen-activated protein kinases (MAPK) (29). In general, these two pathways elicit different responses by the immune system. The MAPK pathway, which is also activated by TNF-α receptors and toll-like receptors (30), has proinflammatory effects (30,31), in part by inducing expression of TNF-α (32). The cAMP pathway, on the contrary, elicits an anti-inflammatory response (33), by decreasing expression of proinflammatory cytokines, such as TNF-α (34) and macrophage inflammatory protein-1 α (35), and by increasing expression of anti-inflammatory cytokines, such as IL-10 (36).
The MC1RR163Q variant, which substitutes a charged arginine residue with a polar glutamine residue in the receptor's GTPase domain (37), is not a simple loss- or gain-of-function mutation. In an in vitro study using human embryonic kidney cells, Doyle et al. (38) found that the MC1RR163Q variant resulted in a loss of MAPK signaling but preserved cAMP signaling. This is distinct from several other MC1R variants (MC1RR151C, MC1RR160W, MC1RD294H) that had normal MAPK activation but attenuated cAMP signalling (29,39). Therefore, the MC1RR163Q variant may lower the risk of a severe systemic response to infection by changing the balance between pro- and anti-inflammatory signals from the MC1R receptor.
It is possible that the MC1RR163Q variant is not causally linked to a lower risk of complicated sepsis; rather the observed association may be due to one of three genetic mechanisms. First, the minor allele frequencies of MC1R SNPs differ greatly between populations (12), so any association between them and a phenotype may be an artifact of population substructure; individuals with the MC1RR163Q variant may have a high degree of common ancestry with many shared variants, any of which may be the true causal variant. We adjusted for population substructure among subjects with available genome-wide data and found that the association was not attenuated, which suggested that the association was not due to population substructure.
Second, other haplotypes may have increased the risk of complicated sepsis. Currently, there is debate about how genetic variants contribute to common complex diseases, with two main hypotheses. The common disease-common variant (CD-CV) hypothesis proposes that many common variants with low penetrance determine genetic susceptibility to common diseases. Alternatively, the common disease-rare variant (CD-RV) hypothesis proposes that multiple rare variants with relatively higher penetrance determine genetic susceptibility to these diseases. Based on the CD-RV hypothesis, it is possible that rare high-risk variants, which we did not test and may have been multiple, were more common in haplotypes that did not contain the MC1RR163Q variant. In this scenario, the MC1RR163Q variant was not responsible for the lower incidence of complicated sepsis, but served as a marker for individuals who did not have a high-risk haplotype. Finally, the MC1RR163Q variant may have tagged a haplotype that included the true causal variant.
The findings from this study have several potential implications. They further elucidate the role of genetic risk in the pathobiology of sepsis. Genotyping has yet to become a component of clinical practice in trauma care, but increasing our understanding of genetic variants has potential to improve risk stratification and influence clinical decisions (40), such as antibiotic use and resuscitation. In addition, if the functional effects of the MC1RR163Q variant are confirmed, it may help direct future drug development. Currently, customized immunomodulatory therapy for sepsis is lacking: recombinant activated protein C, chimeric antibodies against TNF-α, and glucocorticoids have all failed to show a clear survival benefit. Biased agonists that selectively change the signaling pattern of MC1R (41) may be worthwhile alternatives to investigate. An agonist that manipulates MC1R signaling in a manner similar to the MC1RR163Q variant could reduce the risk of a severe inflammatory response after trauma or infection.
This study had several limitations. Like all candidate gene association studies, it may be confounded by population substructure, which was not fully adjusted for in the main analysis. In keeping with the accepted approach to genetic association studies, we restricted our analyses to one race/ethnicity (in this case Caucasians, our largest cohort) so our findings cannot be generalized to other races/ethnicities. Another potential concern is related to the recently updated definitions of sepsis severity by the Sepsis Definitions Task Force, convened by the Society of Critical Care Medicine and the European Society of Intensive Care Medicine (42). According to the new definition, sepsis is an infection with associated organ dysfunction, defined as an acute change in Sequential Organ Failure Assessment (SOFA) score of at least 2 points. SIRS criteria were excluded from the definition because they were neither sensitive nor specific to sepsis. Our current study measured organ dysfunction using the Multiple Organ Dysfunction Score rather than the SOFA score. However, our definition of “complicated sepsis” incorporates both sepsis and septic shock as defined in the Sepsis-3 definitions. Finally, our new finding of an association between MC1R and complicated sepsis requires confirmation in an independent sample of trauma patients before using the gene for screening, risk stratification, or therapeutic targeting.
In conclusion, our study suggested that MC1R has a role in the pathophysiology of sepsis after trauma. The MC1RR163Q variant was associated with a lower risk of complicated sepsis in severely injured trauma patients. This SNP has the potential to be used in “precision medicine” for risk stratification and clinical decision making. Finally, therapeutic targeting of the MC1R pathway may be beneficial for trauma patients who are at high risk for developing complicated sepsis.
The authors thank Alex Reiner, MD, for reviewing the manuscript.
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