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Original Investigation |

The Association of Interleukin 6 Haplotype Clades With Mortality in Critically Ill Adults FREE

Ainsley M. Sutherland, BScH; Keith R. Walley, MD; Sanjay Manocha, MD; James A. Russell, MD
[+] Author Affiliations

Author Affiliations: University of British Columbia McDonald Research Laboratories/The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St Paul’s Hospital, Vancouver, British Columbia.


Arch Intern Med. 2005;165(1):75-82. doi:10.1001/archinte.165.1.75.
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Published online

Background  Interleukin 6 (IL-6) is a key proinflammatory cytokine in the systemic inflammatory response syndrome (SIRS). A G→C polymorphism at position −174 of the IL-6 gene is associated with an adverse outcome in a number of inflammatory diseases, although its association with sepsis as an outcome remains unclear. We tested the hypothesis that specific haplotype clades of IL-6 may be associated with an outcome of SIRS.

Methods  We studied a cohort of 228 critically ill white patients who met at least 2 of 4 SIRS criteria. Clinical data were collected over 28 days after hospital admission. Haplotypes of IL-6 were inferred from publicly available data using PHASE (software for haplotype reconstruction and recombination rate estimation from population data), and cladistic structure was determined using Molecular Evolutionary Genetic Analyses (MEGA2) software. Then, a minimum set of “haplotype tag” single nucleotide polymorphisms (–174G/C, 1753C/G, and 2954G/C) that defined all 4 major haplotype clades of the IL-6 gene was chosen for further genotyping.

Results  Patients who had 2 copies of haplotypes from within the haplotype clades –174C/1753C/2954G (C/C/G), G/G/G, or G/C/C had a greater 28-day mortality compared with patients who carried 1 or no copies of these haplotypes (40.0% vs 26.0%; P = .02). These patients also had fewer days alive and free of multiple system organ dysfunction (P<.05). There were no associations between individual single nucleotide polymorphisms (including –174G/C) and survival or organ dysfunction.

Conclusions  The C/C/G, G/G/G, and G/C/C haplotype clades of IL-6 were strongly associated with increased mortality and more organ dysfunction in a cohort of critically ill patients who had SIRS. Haplotype-based analysis succeeded in identifying this association, whereas individual single nucleotide polymorphism–based analysis failed.

Figures in this Article

A bacterial, viral, or other infection that results in a systemic inflammatory response syndrome (SIRS) is termed sepsis. Sepsis and septic shock have survival rates of approximately 70% and 35% to 45%, respectively.1 Activation of the inflammatory response to infection, including interleukin 6 (IL-6) production, varies significantly between individuals, which is owing in part to genetic variation between individuals. Genotype has been shown to contribute substantially to an outcome of sepsis.24 The genetic contribution to death from sepsis exceeds the inherited genetic contribution to cancer risk of death by many fold and even exceeds the genetic contribution to cardiovascular disease risk.5 Variation in key inflammatory genes such as IL-6 may explain the variation in responses to infection among individuals. This variation has important clinical implications because therapeutic strategies targeting these pathways may be highly effective in patients having specific genotypes, yet it may have adverse effects in other patients with alternative genotypes. It is essential now to test genetic variants for any association with an outcome of complex disease, such as SIRS and sepsis, to incorporate genotype into the design of patient-tailored therapy. Genotype will be used to design therapeutic strategies to optimize outcome while minimizing adverse effects.

Interleukin 6 is a particularly important proinflammatory cytokine in the pathogenesis and outcome of SIRS, sepsis, and septic shock. Increased plasma concentrations of IL-6 are found in patients with sepsis,6,7 and a high IL-6 concentration is associated with increased mortality.8 Furthermore, in related pulmonary inflammation, high plasma concentrations of IL-6 on day 1 of acute respiratory distress syndrome were also associated with increased mortality.9 Therefore, we chose IL-6 as a candidate gene for this association study.

Genetic variation within the IL-6 gene may affect the outcome of sepsis. The C allele of a G→C single nucleotide polymorphism (SNP) at position –174 of the promoter region of the IL-6 gene is associated with decreased levels of IL-6.10 However, one study found there was no association between the –174G/C polymorphism and incidence of sepsis, although –174G homozygosity was associated with improved survival rates in patients with sepsis.11 Thus, the role of the IL-6 genotype in SIRS, sepsis, and septic shock is not clear.

Recently, it has been recognized that single SNP analysis may be less informative than an analysis based on haplotypes. Haplotypes are patterns of several SNPs that are in linkage disequilibrium with one another within a gene or segment of DNA and are thus inherited as a unit (Figure 1).12,13 Single SNP approaches for genetic association studies only work well when the examined SNP is causal or is in significant linkage disequilibrium with the causal SNP. In contrast, haplotypes serve as markers for all measured and unmeasured alleles within the haplotypes.12 A haplotype-based approach can narrow the search for causal SNPs, and haplotypes can serve as predictors of disease severity.12,13 Multiple haplotypes exist for all studied genes; thus, grouping haplotypes into haplotype clades (evolutionarily related groups of haplotypes) increases the statistical power to associate genotype with phenotype.14

Place holder to copy figure label and caption
Figure 1.

Haplotype structure of the interleukin 6 (IL-6) gene. Columns are polymorphic sites. Rows are haplotypes of IL-6 ordered by phylogenetic relationship. Yellow boxes are minor alleles and blue boxes are major alleles. The following were chosen as “haplotype tag” single nucleotide polymorphisms (htSNPs): –174G/C, 1753C/G, and 2954G/C. Halotype clades of IL-6 are indicated by grey, pink, green, and red shading.

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Accordingly, our first aim was to determine whether haplotypes of IL-6 were associated with adverse outcomes in critically ill adults who had SIRS. Our second aim was to determine whether the IL-6 –174G/C polymorphism could account for any association between haplotype and clinical outcome. To accomplish this, we determined the haplotype structure of the IL-6 gene using publicly available data.15 We then used cladistic analysis to group these haplotypes into related clades (Figure 1) and subsequently determined a minimum set of “haplotype tag” SNPs (htSNPs) (Figure 1) that defined all 4 major haplotype clades of the IL-6 gene.14,16,17 We then tested for the association of these haplotype clades with 28-day mortality and organ dysfunction in a cohort of critically ill adults who had SIRS.

PATIENT COHORT

A total of 337 consecutive patients admitted to the tertiary mixed medical-surgical Intensive Care Unit (ICU) of St Paul’s Hospital were screened for inclusion into this study. We only report the results for the white patients (n = 263) to decrease the potential confounding influence of population admixture secondary to ethnic diversity on associations between genotype and phenotype.18 Patient data were screened daily and patients were included in the study cohort (n = 228) if they met at least 2 of 4 SIRS criteria and were successfully genotyped at all 3 htSNPs of IL-6. The SIRS criteria were (1) fever (>38°C) or hypothermia (<36°C), (2) tachycardia (>100 beats/min in the absence of β-blocker therapy), (3) tachypnea (>20 breaths/min) or need for mechanical ventilation, and (4) leukocytosis (total leukocyte count >12 000/μL) or leukopenia (total leukocyte count <4000/μL).1 Patients were included in this cohort on the calendar day that 2 of 4 SIRS criteria were met. This study was approved by the Providence Health Care/University of British Columbia Research Ethics Board.

CLINICAL PHENOTYPE

The primary outcome variable was 28-day mortality. Secondary outcome variables were days alive and free of SIRS, days alive and free of shock, and days alive and free of organ dysfunction (cardiovascular, respiratory, renal, hepatic, hematologic, and neurologic organ systems). Clinically significant organ dysfunction for each organ system was defined as present during a 24-hour period if there was evidence of at least moderate organ dysfunction using the Brussels criteria (Table 1).19

Table Graphic Jump LocationTable 1. Brussels Organ Dysfunction Scoring Criteria*

Because data were not always available during each 24-hour period for each organ dysfunction variable, we used the “carry forward” assumption as defined previously.20 Briefly, for any 24-hour period in which there was no measurement of a variable, we carried forward the measurement from the previous 24-hour period. If any variable was never measured, it was assumed to be normal. Clinical data were recorded for 28 days or until hospital discharge.

To assess duration of organ dysfunction and SIRS and to correct organ dysfunction and SIRS scoring for deaths in the 28-day observation period, we calculated days alive and free of organ dysfunction. Briefly, during each 24-hour period (8 AM to 8 AM) for each variable, days alive and free was scored as 1 if the patient was alive and free of organ dysfunction (normal or mild dysfunction). Days alive and free was scored as 0 if the patient had organ dysfunction (moderate or worse) or was not alive. Every day over the 28-day observation after intensive care unit admission was scored in this way. Thus, the lowest score possible for each variable was zero and the highest score possible was 28. A low score indicates more organ dysfunction, whereas a high score indicates less organ dysfunction.

Baseline demographic data included age, sex, medical vs surgical diagnosis on admission (according to APACHE [Acute Physiology and Chronic Health Evaluation] III diagnostic codes),21 and admission APACHE II score.

HAPLOTYPES AND SELECTION OF htSNPs

We used PHASE (software for haplotype reconstruction and recombination rate estimation from population data) to infer haplotypes of the IL-6 gene from unphased genotypic data from 23 white patients from the Coriell Cell Repository (available at: http://pga.mbt.washington.edu) (Figure 1).15,22 We then used Molecular Evolutionary Genetic Analyses (MEGA2) software to infer a phylogenetic tree to identify major haplotype clades.23 Haplotypes were sorted into clades according to this phylogenetic tree and this haplotype structure was inspected to choose a minimum set of htSNPs (Figure 1).16,17 We chose 3 htSNPs that identified 4 major haplotype clades of IL-6 in white patients. One of these SNPs was the previously reported promoter polymorphism –174G/C (position, National Center for Biotechnology Information [NCBI] ID: 1510G/C, rs1800795). The second SNP was a C→G substitution at nucleotide 1753 relative to the start (3437C/G, rs2069840). The third SNP was a G→C substitution at nucleotide 2954 (4638G/C, rs1548216) (NCBI IL-6 accession number AF372214). These 3 SNPs were then genotyped in our 228 patient cohort and PHASE software was used to identify haplotypes in each patient.

GENOTYPING

Patients’ genotypes at positions 1753 and 2954 were determined by real-time polymerase chain reaction assay using specific fluorescence-labeled hybridization probes in the ABI Prism 7900HT Sequence Detection System (Applied Biosystems Inc, Foster City, Calif).24 DNA with known genotype from 23 lymphocyte cell lines from the Coriell Cell Repository demonstrated complete concordance with our genotyping at positions 1753 and 2954 of the IL-6 gene.15 Genotyping of –174G/C was performed using both a previously reported polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method and a matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF) approach (Qiagen Inc, Mississauga, Ontario), which again demonstrated complete concordance.25,26

STATISTICAL ANALYSIS

We used a cohort study design. Rates of dichotomous outcomes (28-day mortality, sepsis and shock at onset of SIRS) were compared between clades. The C/C/G, G/G/G, and G/C/C clades appeared to be associated with increased 28-day mortality and organ dysfunction. Further comparisons were then made between patients carrying 2 haplotypes from within the C/C/G, G/G/G, or G/C/C haplotype clades and patients carrying only 1 or no copies of a haplotype from within the C/C/G, G/G/G, or G/C/C haplotype clades using a χ2 test, assuming a recessive model of inheritance. Differences in continuous outcome variables between patients with 2 haplotypes from within the haplotype clades C/C/G, G/G/G, or G/C/C and patients carrying only 1 or no copies of a haplotype from within the C/C/G, G/G/G, or G/C/C haplotype clades were tested by analysis of variance. A Cox regression analysis as well as a Kaplan-Meier analysis of censored survival data was used to further compare 28-day mortality between the 2 groups of patients while adjusting for other confounders (age, sex, and medical vs surgical diagnosis). Haplotype clade relative risk was calculated. Genotype distributions were tested for Hardy-Weinberg equilibrium.27 We report the mean and 95% confidence intervals. Statistical significance was set at P<.05. The data were analyzed using SPSS 11.5 for Windows (SPSS Inc, Chicago, Ill).

We were able to infer haplotypes from complete sequencing of IL-6 for 23 white patients in the Coriell Cell Repository using PHASE software and identified 4 major haplotype clades using MEGA2 software (Figure 1).22,23 These 4 clades could be resolved by genotyping 3 htSNPs (–174G/C, 1753C/G, and 2954G/C) in our 228 patient cohort.16 The –174C/1753C/2954G (C/C/G) haplotype clade occurred with a frequency of 43.2%; the G/G/G haplotype clade, 32.9%; the G/C/G haplotype clade, 20.6%; and the G/C/C haplotype clade, 3.1%. A fifth haplotype defined by –174C/1753G/2954G occurred only once (0.2%) and thus was not included in further presentation of these data. These frequencies were similar to frequencies deduced from other available data on white patients.15

For the 228 successfully genotyped individuals of the cohort of white patients who met at least 2 of 4 SIRS criteria, no haplotype clade of IL-6 was significantly associated with a difference in age, sex, medical vs surgical diagnoses at admission, or severity of illness at the time of admission (as estimated by the APACHE II score) (Table 2).

Table Graphic Jump LocationTable 2. Baseline Characteristics of 228 Critically Ill Adults With SIRS

The IL-6 haplotype clades C/C/G, G/G/G, and G/C/C were associated with greater 28-day mortality rates than the G/C/G haplotype clade. Patients who carried any combination of 2 haplotypes from within the haplotype clades C/C/G, G/G/G, or G/C/C had significantly greater 28-day mortality compared with patients who carried at least 1 copy of the G/C/G haplotype clade (P = .02) (Figure 2). Carrying 2 copies of the C/C/G, G/G/G, or G/C/C haplotype clades conferred a relative risk of mortality of 1.27. After adjusting for other predictors of survival (age, sex, medical vs surgical diagnosis at admission) using a Cox regression analysis, patients carrying 2 haplotypes from within the haplotype clades C/C/G, G/G/G, or G/C/C still had significantly greater rates of 28-day mortality with a hazard ratio of 1.83 (P = .02) (Table 3). Kaplan-Meier analysis of censored 28-day survival data confirmed that patients carrying 2 copies of the C/C/G, G/G/G, or G/C/C haplotype clades had increased probability of mortality during the 28-day observation period (P = .01) (Figure 3).

Place holder to copy figure label and caption
Figure 2.

Twenty-eight day mortality rates by interleukin 6 (IL-6) clade. A, The G/C/G haplotype clade was associated with decreased 28-day mortality (P = .02). B, Critically ill patients with 2 copies of the C/C/G, G/G/G, or G/C/C haplotype clades had greater 28-day mortality than those with 1 or no copies (P = .02). The C/C/G, G/G/G, or G/C/C bar is clear to highlight the significant difference in mortality. Error bars represent 95% confidence intervals.

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Place holder to copy figure label and caption
Figure 3.

Kaplan-Meier mortality analysis by interleukin 6 (IL-6) clade. Kaplan-Meier analysis showed that patients with 2 copies of the C/C/G, G/G/G, or G/C/C haplotype clades had greater mortality rates compared with patients with at least 1 copy of the G/C/G haplotype clade for the entire 28-day observation period (P = .01).

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Table Graphic Jump LocationTable 3. Cox Proportional Hazard Analysis—Hazard Ratios for Mortality

Patients carrying 2 copies of the C/C/G, G/G/G, or G/C/C haplotype clades had significantly fewer days alive and free of cardiovascular dysfunction (Figure 4) (P = .006) and required more cardiovascular support as measured by fewer days alive and free of vasopressors (Figure 4) (P = .01). Patients carrying 2 copies of the C/C/G, G/G/G, or G/C/C haplotype clades also had significantly fewer days alive and free of acute lung injury (Figure 4) (P = .002) and fewer days alive and free of ventilation (Figure 4) (P = .04). The C/C/G, G/G/G, and G/C/C haplotype clades were associated with fewer days alive and free of renal dysfunction (Figure 4) (P = .005) and a trend to fewer days alive and free of renal support (Figure 4) (P = .07). Furthermore, patients who carried 2 copies of the C/C/G, G/G/G, or G/C/C haplotype clades had significantly more hematologic system dysfunction (18.7 days alive and free of hematologic dysfunction for patients with 2 copies of C/C/G, G/G/G, or G/C/C vs 21.0 days for patients with 1 or no copies; P = .02).

Place holder to copy figure label and caption
Figure 4.

Multiple-system organ dysfunction in critically ill patients. Patients with 2 copies of the C/C/G, G/G/G, or G/C/C clades had fewer days alive and free of cardiovascular system (CVS) dysfunction (P = .006) and vasopressors (P = .01) (A), fewer days alive and free of acute lung injury (ALI) (P = .002) and ventilation (P = .04) (B), and fewer days alive and free of renal dysfunction (P = .005) (C).

Graphic Jump Location

Patients who carried 2 copies of the C/C/G, G/G/G, or G/C/C haplotype clades had significantly fewer days alive and free of 4 of 4 (18.4 days alive and free vs 21.1 days; P = .03) and 3 of 4 SIRS criteria (13.9 days alive and free vs 17.4 days; P = .006) compared with patients who carried 1 or no copies of the C/C/G, G/G/G, or G/C/C haplotype clades.

In contrast to the strong association between the IL-6 C/C/G, G/G/G, and G/C/C haplotype clades with adverse clinical outcome, there was no association between any of the individual SNPs and the clinical outcome variables. Specifically, there was no association between −174G/C and increased 28-day mortality. There was also no significant association between the –174G/C polymorphism and cardiovascular, hepatic, hematologic, or neurologic system dysfunction. The genotype distribution of –174G/C was similar to the frequencies reported in other critically ill white patients (Table 4).11 The sample population was in Hardy-Weinberg equilibrium for all genotyped SNPs.27

Table Graphic Jump LocationTable 4. Genotype Frequencies and Allele Frequencies for Interleukin 6 htSNPs –174G/C, 1753C/G, and 2954G/C in 228 Critically Ill Adults With SIRS

We found that the C/C/G, G/G/G, and G/C/C haplotype clades of IL-6 were strongly and significantly associated with increased 28-day mortality and increased organ dysfunction in critically ill white patients who had SIRS. Because none of the individual htSNPs used to define the haplotype clades of IL-6 were associated with survival, haplotype analysis of IL-6 proved to be a more valuable tool in identifying genetic associations compared with individual SNP analysis. These findings further support a pivotal role of IL-6 in the pathogenesis of the systemic inflammatory response syndrome.

Identification of important associations between genotype and clinical outcome is important for clinicians caring for critically ill patients for 2 key reasons. First, there is a substantial genetic contribution to the degree of activation of the systemic inflammatory response in patients with sepsis. The association of a gene polymorphism with outcome implicates pathways involving that gene in the pathophysiologic mechanisms of the clinical outcome—and thereby identifies potential therapeutic targets. Second, and more important, the benefit of specific therapy will depend on genotype. For example, anti-inflammatory therapy is a double-edged sword globally modulating both appropriate and inappropriate inflammatory responses. Thus, a number of recently developed immunomodulatory therapies will be beneficial in patients with specific genotypes and not beneficial in others. This view is consistent with the observations that indiscriminate application (by genotype) of many promising immunomodulatory therapies failed in phase 3 randomized controlled trials. It will soon become technologically feasible to screen patients at the bedside to predict this response and design patient-tailored therapy using markers of clinical outcome such as the IL-6 C/C/G, G/G/G, and G/C/C haplotype clades. Already there are a number of high-throughput DNA chips being developed for rapid bedside genotyping of clinically relevant polymorphisms.28,29 Genetic information will be used by the clinician to define clinical subtypes of disease and to stratify patients according to their risk of poor outcome. Genotype will be used to determine the optimal drug and its dose for treatment of an individual while minimizing adverse effects. This knowledge will be instrumental in both preventative medicine and treatment decisions.

Analysis of haplotypes of SNPs within a gene is a powerful approach to study the association of common genetic variants with susceptibility to common diseases.12 Haplotype analysis is more effective in determining association of genotype with phenotype than is individual SNP analysis.12,13 Haplotypes serve as markers of unidentified polymorphisms that may be the cause of phenotypic variation.12 The phylogenetic history of haplotypes within a population may be determined and used to group haplotypes into clades using cladistic analysis.14,3033 Cladistic analysis has 2 unique strengths. First, grouping haplotypes into clades decreases the degrees of freedom, thereby increasing the statistical power to associate genotype with phenotype.13 Second, grouping haplotypes into clades facilitates identification of causal SNPs. Another practical strength of our approach is that a small number of htSNPs can be used to distinguish haplotype clades, eliminating the need to genotype all SNPs within a gene.16,34 We selected as htSNPs the previously reported SNP –174G/C, as well as 1753C/G and 2954G/C, to define the 4 major haplotype clades within the IL-6 gene.

To our knowledge, this is the first report of a significant association of the IL-6 C/C/G, G/G/G, and G/C/C haplotype clades with 28-day mortality in critically ill patients with SIRS. None of the htSNPs were individually associated with increased 28-day mortality individually, demonstrating the power and utility of haplotypes in association studies of genotype with phenotype. The SNP that is responsible for the deleterious effect of the C/C/G, G/G/G, and G/C/C haplotype clades in patients with SIRS is most likely an SNP that is common to these 3 clades and different in the G/C/G haplotype clade. Thus, by using a haplotype-based approach to associate genetic variation within the IL-6 gene with survival of critically ill patients, we substantially narrowed down the number of SNPs in the IL-6 gene that may cause increased mortality in patients with sepsis. Alternatively, an SNP within the G/C/G halotype clade may have a beneficial effect that improves outcome of SIRS.

Our finding of an association of the IL-6 C/C/G, G/G/G, and G/C/C haplotype clades with increased mortality in critically ill patients was strengthened by discovering significant associations of the C/C/G, G/G/G, and G/C/C haplotype clades with secondary clinical outcomes. Patients who had 2 copies of the C/C/G, G/G/G, and G/C/C haplotype clades had a more severe systemic inflammatory response (fewer days alive and free of SIRS). Patients carrying 2 copies of the detrimental C/C/G, G/G/G, and G/C/C haplotype clades also had fewer days alive and free of other organ dysfunction. These findings amplify the harmful association of the IL-6 C/C/G, G/G/G, and G/C/C haplotype clades with mortality in SIRS.

Previous studies of the association of polymorphisms of IL-6 with outcomes of inflammatory diseases yielded conflicting results. Fishman et al10 demonstrated that a promoter region construct containing the C allele of the IL-6 –174G/C polymorphism transiently transfected into HeLa cells yielded lower expression of IL-6 than the –174G construct, both at baseline and after stimulation with lipopolysaccharide or IL-1. The C allele was also associated with less systemic-onset juvenile chronic arthritis.10 The IL-6 –174C allele has been associated with increased survival rates of patients with adult respiratory distress syndrome.35 GG homozygosity at –174G/C has also been associated with greater plasma concentrations of IL-6 and greater duration of intensive care unit and hospital stay following coronary artery bypass graft surgery.36 In contrast, there is no association of IL-6 –174G/C genotype with systemic lupus erythematosus,37 incidence of sepsis,11 or with the IL-6 response to endotoxin in whole blood from patients with sepsis.38,39 Surprisingly, the G allele was associated with improved survival in patients with sepsis.11 Because –174G/C was one of our htSNPs, our results directly address these conflicting results. We found no association of –174 G or C alleles with outcome in this critically ill SIRS population. However, genotype of this SNP contributes to defining the IL-6 C/C/G, G/G/G, and G/C/C haplotype clades that were associated with strikingly poorer outcome.

Limitations of genetic association studies should be addressed. First, our large cohort of critically ill patients (n = 228) reduced the risk of type I error (finding a spurious association) compared with other studies of smaller sample size (n = 20-100).4043 Second, ethnic heterogeneity within a study population can also lead to false-positive associations between genotype and phenotype.18 Therefore, we included only white patients in our cohort of critically ill adults to decrease the risk of positive associations due to population stratification. Third, we further limited type I error by using several secondary measures of clinical phenotype in addition to the primary outcome variable. Thus, our association of the C/C/G, G/G/G, and G/C/C haplotype clades with increased 28-day mortality in critically ill patients was supported by the association of the C/C/G, G/G/G, and G/C/C haplotype clades with worsened secondary outcome variables. Furthermore, the association of the C/C/G, G/G/G, and G/C/C haplotype clades with multiple worsened secondary outcomes was also consistent with the association of the C/C/G, G/G/G, and G/C/C haplotype clades with increased need for organ life support.

Statistical power is important in genetic association studies. By grouping haplotypes into clades to test for association with clinical outcomes, we decreased degrees of freedom and increased statistical power to detect associations.13 We used strategies to maximize the accuracy of the inferred haplotypes of IL-6. We used PHASE software to infer haplotypes of the IL-6 gene from unphased genotypic data from white patients (available at: http://pga.mbt.washington.edu).15,22 We did not have access to the genotypes of family members of the genotyped individuals, and therefore could only infer probable haplotypes of the IL-6 gene using statistical methods.22 Importantly, Stephens et al22 have shown that PHASE is highly accurate. Therefore, reconstruction of haplotypes experimentally or by genotyping additional family members may not provide further information.22 Furthermore, PHASE software provides estimates of the certainty of haplotype assignment. In view of the fairly simple haplotype structure of the IL-6 gene, the PHASE algorithm determined with absolute certainty 98% of the phase calls in our cohort, and estimated the certainty associated with the remaining 2% of phase calls to be 0.99.

A limitation of haplotype association studies is that association of the C/C/G, G/G/G, and G/C/C haplotype clades with poor outcome does not identify an injurious SNP but importantly narrows the possibilities.12 The association of haplotypes of IL-6 with increased mortality does not provide us with a pathological mechanism for the increase in mortality. Given that the injurious SNP has not been identified, our results do not address how the C/C/G, G/G/G, and G/C/C haplotype clades affect regulation and/or levels of IL-6. In addition, because we have not measured RNA expression or serum levels of IL-6 in our cohort of critically ill patients, we are not able to conclude what functional consequences haplotypes of IL-6 may have in sepsis and the inflammatory response. However, we stress that the C/C/G, G/G/G, and G/C/C haplotype clades are an accurate marker of the enhanced chance of poor outcome of SIRS.

In summary, the G→C polymorphism at position –174 is not associated with outcome in patients who have SIRS. In contrast, the C/C/G, G/G/G, and G/C/C haplotype clades were significantly associated with increased 28-day mortality, fewer days alive and free of SIRS, and fewer days alive and free of multisystem organ dysfunction in a cohort of critically ill white patients who had SIRS. This is further evidence of a pivotal role for IL-6 in the pathologic process of SIRS.

Correspondence: James A. Russell, MD, St Paul’s Hospital, 1081 Burrard St, Vancouver, British Columbia, Canada V6Z 1Y6 (jrussell@providencehealth.bc.ca).

Accepted for Publication: July 20, 2004.

Financial Disclosure: None.

Funding/Support: This study was supported by the Canadian Institutes of Health Research, Ottawa, Ontario.

Previous Presentation: This study was presented in part at the American Thoracic Society International Conference; May 18, 2003; Seattle, Wash.

Additional Information: Dr Walley is a Michael Smith Foundation for Health Research Distinguished Scholar.

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Zhang  KCalabrese  PNordborg  MSun  F Haplotype block structure and its applications to association studies: power and study designs. Am J Hum Genet 2002;711386- 1394
PubMed Link to Article
Templeton  ARWeiss  KMNickerson  DABoerwinkle  ESing  CF Cladistic structure within the human lipoprotein lipase gene and its implications for phenotypic association studies. Genetics 2000;1561259- 1275
PubMed
Rieder  MJCarrington  DPChung  M-WLee  KLPoel  CLYi  QNickerson  DA Homo sapiens interleukin 6 (IL6) gene, complete cds. ACCESSION AF372214 2002 vol: SeattleSNPs Seattle, Wash NHLBI Program for Genomic ApplicationsUW-FHCRC; 2001. Available at: http://pga.mbt.washington.edu
Johnson  GCEsposito  LBarratt  BJ  et al.  Haplotype tagging for the identification of common disease genes. Nat Genet 2001;29233- 237
PubMed Link to Article
Gabriel  SBSchaffner  SFNguyen  H  et al.  The structure of haplotype blocks in the human genome. Science 2002;2962225- 2229
PubMed Link to Article
Cardon  LRBell  JI Association study designs for complex diseases. Nat Rev Genet 2001;291- 99
PubMed Link to Article
Sibbald  WJVincent  JL Round table conference on clinical trials for the treatment of sepsis: Brussels, March 12-14, 1994. Intensive Care Med 1995;21184- 189
PubMed Link to Article
Bernard  GRWheeler  APArons  MM  et al. The Antioxidant in ARDS Study Group, A trial of antioxidants N-acetylcysteine and procysteine in ARDS. Chest 1997;112164- 172
PubMed Link to Article
Knaus  WAWagner  DPDraper  EA  et al.  The APACHE III prognostic system. Risk prediction of hospital mortality for critically ill hospitalized adults. Chest 1991;1001619- 1636
PubMed Link to Article
Stephens  MSmith  NJDonnelly  P A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 2001;68978- 989
PubMed Link to Article
Kumar  STamura  KJakobsen  IBNei  M MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 2001;171244- 1245
PubMed Link to Article
Livak  KJ Allelic discrimination using fluorogenic probes and the 5′ nuclease assay. Genet Anal 1999;14143- 149
PubMed Link to Article
Pascual  MNieto  AMataran  LBalsa  APascual-Salcedo  DMartin  J IL-6 promoter polymorphisms in rheumatoid arthritis. Genes Immun 2000;1338- 340
PubMed Link to Article
Bray  MSBoerwinkle  EDoris  PA High-throughput multiplex SNP genotyping with MALDI-TOF mass spectrometry: practice, problems and promise. Hum Mutat 2001;17296- 304
PubMed Link to Article
Guo  SWThompson  EA Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 1992;48361- 372
PubMed Link to Article
Zhong  XBReynolds  RKidd  JR  et al.  Single-nucleotide polymorphism genotyping on optical thin-film biosensor chips. Proc Natl Acad Sci U S A 2003;10011559- 11564
Link to Article
Nakai  KHabano  WFujita  T  et al.  Highly multiplexed genotyping of coronary artery disease-associated SNPs using MALDI-TOF mass spectrometry. Hum Mutat 2002;20133- 138
PubMed Link to Article
Templeton  ARCrandall  KASing  CF A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data, III: cladogram estimation. Genetics 1992;132619- 633
PubMed
Templeton  AR A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping or DNA sequencing, V: analysis of case/control sampling designs: Alzheimer’s disease and the apoprotein E locus. Genetics 1995;140403- 409
PubMed
Long  ADLangley  CH The power of association studies to detect the contribution of candidate genetic loci to variation in complex traits. Genome Res 1999;9720- 731
PubMed
Braaten  ORodningen  OKNordal  ILeren  TP The genetic algorithm applied to haplotype data at the LDL receptor locus. Comput Methods Programs Biomed 2000;611- 9
PubMed Link to Article
Weiss  KMClark  AG Linkage disequilibrium and the mapping of complex human traits. Trends Genet 2002;1819- 24
PubMed Link to Article
Marshall  RPWebb  SHill  MRHumphries  SELaurent  GJ Genetic polymorphisms associated with susceptibility and outcome in ARDS. Chest 2002;12168S- 69S
PubMed Link to Article
Burzotta  FIacoviello  LDi Castelnuovo  A  et al.  Relation of the −174G/C polymorphism of interleukin-6 to interleukin-6 plasma levels and to length of hospitalization after surgical coronary revascularization. Am J Cardiol 2001;881125- 1128
PubMed Link to Article
Linker-Israeli  MWallace  DJPrehn  J  et al.  Association of IL-6 gene alleles with systemic lupus erythematosus (SLE) and with elevated IL-6 expression. Genes Immun 1999;145- 52
PubMed Link to Article
Heesen  MBloemeke  BHeussen  NKunz  D Can the interleukin-6 response to endotoxin be predicted? studies of the influence of a promoter polymorphism of the interleukin-6 gene, gender, the density of the endotoxin receptor CD14, and inflammatory cytokines. Crit Care Med 2002;30664- 669
PubMed Link to Article
Heesen  MObertacke  USchade  FUBloemeke  BMajetschak  M The interleukin-6 G(-174)C polymorphism and the ex vivo interleukin-6 response to endotoxin in severely injured blunt trauma patients. Eur Cytokine Netw 2002;1372- 77
PubMed
Arnalich  FLopez-Maderuelo  DCodoceo  R  et al.  Interleukin-1 receptor antagonist gene polymorphism and mortality in patients with severe sepsis. Clin Exp Immunol 2002;127331- 336
PubMed Link to Article
Gibot  SCariou  ADrouet  LRossignol  MRipoll  L Association between a genomic polymorphism within the CD14 locus and septic shock susceptibility and mortality rate. Crit Care Med 2002;30969- 973
PubMed Link to Article
Lowe  PRGalley  HFAbdel-Fattah  AWebster  NR Influence of interleukin-10 polymorphisms on interleukin-10 expression and survival in critically ill patients. Crit Care Med 2003;3134- 38
PubMed Link to Article
Schaaf  BMBoehmke  FEsnaashari  H  et al.  Pneumococcal septic shock is associated with the interleukin-10-1082 gene promoter polymorphism. Am J Respir Crit Care Med 2003;168476- 480
PubMed Link to Article

Figures

Place holder to copy figure label and caption
Figure 1.

Haplotype structure of the interleukin 6 (IL-6) gene. Columns are polymorphic sites. Rows are haplotypes of IL-6 ordered by phylogenetic relationship. Yellow boxes are minor alleles and blue boxes are major alleles. The following were chosen as “haplotype tag” single nucleotide polymorphisms (htSNPs): –174G/C, 1753C/G, and 2954G/C. Halotype clades of IL-6 are indicated by grey, pink, green, and red shading.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Twenty-eight day mortality rates by interleukin 6 (IL-6) clade. A, The G/C/G haplotype clade was associated with decreased 28-day mortality (P = .02). B, Critically ill patients with 2 copies of the C/C/G, G/G/G, or G/C/C haplotype clades had greater 28-day mortality than those with 1 or no copies (P = .02). The C/C/G, G/G/G, or G/C/C bar is clear to highlight the significant difference in mortality. Error bars represent 95% confidence intervals.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Kaplan-Meier mortality analysis by interleukin 6 (IL-6) clade. Kaplan-Meier analysis showed that patients with 2 copies of the C/C/G, G/G/G, or G/C/C haplotype clades had greater mortality rates compared with patients with at least 1 copy of the G/C/G haplotype clade for the entire 28-day observation period (P = .01).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Multiple-system organ dysfunction in critically ill patients. Patients with 2 copies of the C/C/G, G/G/G, or G/C/C clades had fewer days alive and free of cardiovascular system (CVS) dysfunction (P = .006) and vasopressors (P = .01) (A), fewer days alive and free of acute lung injury (ALI) (P = .002) and ventilation (P = .04) (B), and fewer days alive and free of renal dysfunction (P = .005) (C).

Graphic Jump Location

Tables

Table Graphic Jump LocationTable 1. Brussels Organ Dysfunction Scoring Criteria*
Table Graphic Jump LocationTable 2. Baseline Characteristics of 228 Critically Ill Adults With SIRS
Table Graphic Jump LocationTable 3. Cox Proportional Hazard Analysis—Hazard Ratios for Mortality
Table Graphic Jump LocationTable 4. Genotype Frequencies and Allele Frequencies for Interleukin 6 htSNPs –174G/C, 1753C/G, and 2954G/C in 228 Critically Ill Adults With SIRS

References

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PubMed Link to Article
Majetschak  MFlohe  SObertacke  U  et al.  Relation of a TNF gene polymorphism to severe sepsis in trauma patients. Ann Surg 1999;230207- 214
PubMed Link to Article
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PubMed Link to Article
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PubMed Link to Article
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PubMed
Waage  ABrandtzaeg  PHalstensen  AKierulf  PEspevik  T The complex pattern of cytokines in serum from patients with meningococcal septic shock: association between interleukin 6, interleukin 1, and fatal outcome. J Exp Med 1989;169333- 338
PubMed Link to Article
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PubMed Link to Article
Meduri  GUHeadley  SKohler  G  et al.  Persistent elevation of inflammatory cytokines predicts a poor outcome in ARDS: plasma IL-1 beta and IL-6 levels are consistent and efficient predictors of outcome over time. Chest 1995;1071062- 1073
PubMed Link to Article
Fishman  DFaulds  GJeffery  R  et al.  The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 1998;1021369- 1376
PubMed Link to Article
Schluter  BRaufhake  CErren  M  et al.  Effect of the interleukin-6 promoter polymorphism (-174 G/C) on the incidence and outcome of sepsis. Crit Care Med 2002;3032- 37
PubMed Link to Article
Akey  JJin  LXiong  M Haplotypes vs single marker linkage disequilibrium tests: what do we gain? Eur J Hum Genet 2001;9291- 300
PubMed Link to Article
Zhang  KCalabrese  PNordborg  MSun  F Haplotype block structure and its applications to association studies: power and study designs. Am J Hum Genet 2002;711386- 1394
PubMed Link to Article
Templeton  ARWeiss  KMNickerson  DABoerwinkle  ESing  CF Cladistic structure within the human lipoprotein lipase gene and its implications for phenotypic association studies. Genetics 2000;1561259- 1275
PubMed
Rieder  MJCarrington  DPChung  M-WLee  KLPoel  CLYi  QNickerson  DA Homo sapiens interleukin 6 (IL6) gene, complete cds. ACCESSION AF372214 2002 vol: SeattleSNPs Seattle, Wash NHLBI Program for Genomic ApplicationsUW-FHCRC; 2001. Available at: http://pga.mbt.washington.edu
Johnson  GCEsposito  LBarratt  BJ  et al.  Haplotype tagging for the identification of common disease genes. Nat Genet 2001;29233- 237
PubMed Link to Article
Gabriel  SBSchaffner  SFNguyen  H  et al.  The structure of haplotype blocks in the human genome. Science 2002;2962225- 2229
PubMed Link to Article
Cardon  LRBell  JI Association study designs for complex diseases. Nat Rev Genet 2001;291- 99
PubMed Link to Article
Sibbald  WJVincent  JL Round table conference on clinical trials for the treatment of sepsis: Brussels, March 12-14, 1994. Intensive Care Med 1995;21184- 189
PubMed Link to Article
Bernard  GRWheeler  APArons  MM  et al. The Antioxidant in ARDS Study Group, A trial of antioxidants N-acetylcysteine and procysteine in ARDS. Chest 1997;112164- 172
PubMed Link to Article
Knaus  WAWagner  DPDraper  EA  et al.  The APACHE III prognostic system. Risk prediction of hospital mortality for critically ill hospitalized adults. Chest 1991;1001619- 1636
PubMed Link to Article
Stephens  MSmith  NJDonnelly  P A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 2001;68978- 989
PubMed Link to Article
Kumar  STamura  KJakobsen  IBNei  M MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 2001;171244- 1245
PubMed Link to Article
Livak  KJ Allelic discrimination using fluorogenic probes and the 5′ nuclease assay. Genet Anal 1999;14143- 149
PubMed Link to Article
Pascual  MNieto  AMataran  LBalsa  APascual-Salcedo  DMartin  J IL-6 promoter polymorphisms in rheumatoid arthritis. Genes Immun 2000;1338- 340
PubMed Link to Article
Bray  MSBoerwinkle  EDoris  PA High-throughput multiplex SNP genotyping with MALDI-TOF mass spectrometry: practice, problems and promise. Hum Mutat 2001;17296- 304
PubMed Link to Article
Guo  SWThompson  EA Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 1992;48361- 372
PubMed Link to Article
Zhong  XBReynolds  RKidd  JR  et al.  Single-nucleotide polymorphism genotyping on optical thin-film biosensor chips. Proc Natl Acad Sci U S A 2003;10011559- 11564
Link to Article
Nakai  KHabano  WFujita  T  et al.  Highly multiplexed genotyping of coronary artery disease-associated SNPs using MALDI-TOF mass spectrometry. Hum Mutat 2002;20133- 138
PubMed Link to Article
Templeton  ARCrandall  KASing  CF A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data, III: cladogram estimation. Genetics 1992;132619- 633
PubMed
Templeton  AR A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping or DNA sequencing, V: analysis of case/control sampling designs: Alzheimer’s disease and the apoprotein E locus. Genetics 1995;140403- 409
PubMed
Long  ADLangley  CH The power of association studies to detect the contribution of candidate genetic loci to variation in complex traits. Genome Res 1999;9720- 731
PubMed
Braaten  ORodningen  OKNordal  ILeren  TP The genetic algorithm applied to haplotype data at the LDL receptor locus. Comput Methods Programs Biomed 2000;611- 9
PubMed Link to Article
Weiss  KMClark  AG Linkage disequilibrium and the mapping of complex human traits. Trends Genet 2002;1819- 24
PubMed Link to Article
Marshall  RPWebb  SHill  MRHumphries  SELaurent  GJ Genetic polymorphisms associated with susceptibility and outcome in ARDS. Chest 2002;12168S- 69S
PubMed Link to Article
Burzotta  FIacoviello  LDi Castelnuovo  A  et al.  Relation of the −174G/C polymorphism of interleukin-6 to interleukin-6 plasma levels and to length of hospitalization after surgical coronary revascularization. Am J Cardiol 2001;881125- 1128
PubMed Link to Article
Linker-Israeli  MWallace  DJPrehn  J  et al.  Association of IL-6 gene alleles with systemic lupus erythematosus (SLE) and with elevated IL-6 expression. Genes Immun 1999;145- 52
PubMed Link to Article
Heesen  MBloemeke  BHeussen  NKunz  D Can the interleukin-6 response to endotoxin be predicted? studies of the influence of a promoter polymorphism of the interleukin-6 gene, gender, the density of the endotoxin receptor CD14, and inflammatory cytokines. Crit Care Med 2002;30664- 669
PubMed Link to Article
Heesen  MObertacke  USchade  FUBloemeke  BMajetschak  M The interleukin-6 G(-174)C polymorphism and the ex vivo interleukin-6 response to endotoxin in severely injured blunt trauma patients. Eur Cytokine Netw 2002;1372- 77
PubMed
Arnalich  FLopez-Maderuelo  DCodoceo  R  et al.  Interleukin-1 receptor antagonist gene polymorphism and mortality in patients with severe sepsis. Clin Exp Immunol 2002;127331- 336
PubMed Link to Article
Gibot  SCariou  ADrouet  LRossignol  MRipoll  L Association between a genomic polymorphism within the CD14 locus and septic shock susceptibility and mortality rate. Crit Care Med 2002;30969- 973
PubMed Link to Article
Lowe  PRGalley  HFAbdel-Fattah  AWebster  NR Influence of interleukin-10 polymorphisms on interleukin-10 expression and survival in critically ill patients. Crit Care Med 2003;3134- 38
PubMed Link to Article
Schaaf  BMBoehmke  FEsnaashari  H  et al.  Pneumococcal septic shock is associated with the interleukin-10-1082 gene promoter polymorphism. Am J Respir Crit Care Med 2003;168476- 480
PubMed Link to Article

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