Inflammation is strongly and independently associated with cardiovascular disease (CVD) risk. It may explain the excess CVD risk in dialysis patients, in whom inflammation is common (1). Interleukin-6 (IL-6) is a major proinflammatory cytokine that is central to the inflammatory response, regulating the hepatic synthesis of acute-phase proteins, such as fibrinogen, C-reactive protein, and albumin. IL-6 mRNA is present in atherosclerotic arteries at a 10- to 40-fold higher level than in nonatherosclerotic vessels, and elevated levels of IL-6 are associated with increased risk for CVD, suggesting that IL6 has a role in the pathogenesis of atherosclerosis (2,3). Whether the relationship of IL-6 with CVD is causal, however, is difficult to prove because atherosclerosis also may induce the synthesis of inflammatory markers.
Establishing a role of genetic variants in susceptibility to CVD would bolster the inference that high levels of the inflammatory response to environmental stimuli can lead to CVD, because genetic variants cannot be a consequence of CVD. More light has been shed on a common G/C polymorphism at position −174 in the promoter region of the IL6 gene. The −174C allele has been associated with higher risk for CVD incidence and mortality in some studies of white patients (4–7) but not in others (8,9). Circulating concentrations of IL-6 are thought to be largely regulated at the level of expression; however, the role of the −174G/C variant is uncertain (10–12). Moreover, two missense variants in the coding region have been reported (13) but have not yet been examined in an association study.
The high variability in levels of inflammatory markers along with the very high rate of CVD in dialysis patients may allow the determination of subtle effects of the IL6 gene. Also, allelic heterogeneity should be considered because individual haplotypes may have differential effects (12). We investigated the influence of potential functional variants and common haplotypes in the IL6 gene on levels of gene expression, inflammatory marker, and CVD risk in a prospective study of dialysis patients.
Materials and Methods
Study Design and Population
The CHOICE study is a national prospective cohort study of 1041 incident dialysis patients who are aged 19 to 95 yr and from 81 dialysis clinics. The study was approved by the Institutional Review Boards, and participants provided written informed consent. The study design and enrollment criteria have been described elsewhere (14). Participants were enrolled from October 1995 to June 1998, median of 45 d after initiation of dialysis (95% within 3.5 mo), and were followed up through November 2000. Genotype information was available on 775 of 898 participants for whom blood was drawn before a dialysis session at a median of 5.0 mo from the initiation of dialysis (95% within 8.7 mo). Their baseline demographic and clinical data were obtained from questionnaires, as well as from hospital and clinic records. Prevalent CVD was defined as medical record documentation of coronary artery disease, cerebrovascular disease, or peripheral vascular disease. The level of cardiovascular and other comorbidity was assessed by a trained nurse on the basis of medical records and clinic staff reports using the Index of Co-Existent Disease, a standardized and validated four-level scale that has been tested in multiple studies (15).
DNA Analysis
The resequencing data of 24 African-American and 23 European patients from the Program for Genomic Applications at the University of Washington (13) were used to choose sequence variants to genotype. On the basis of 31 retrieved variants in the IL6 gene, 11 polymorphisms were selected in this study, including three previously described polymorphisms, −174G/C, −572G/C, and −597G/A, and two novel coding variants, Pro32Ser (C>T) and Asp162Val (A>T), along with six other polymorphisms (rs2069825, rs2069827, rs2069840, rs1554606, rs2069845, and rs2069849), which differentiate the 10 common haplotypes (Figure 1). Haplotypes were constructed separately for black and white patient using the PHASE program, version 1.0.1 (16).
Because of a limited statistical power to detect effects of the multiple haplotypes and the multiple comparison issues, we sought a method to classify haplotypes. Cladistic method, in which the evolutionary history of the haplotype variation is estimated, has been proposed for phenotypic association study (17,18). Such an approach requires that the cladistic structure not be disrupted by recombination and can be estimated. In the IL6 gene, most of the variants are in linkage disequilibrium and belong to a single haplotype block (defined using the Confidence Interval Method [19] implemented in Haploview 3.2). The cladistic approach, therefore, was chosen for grouping haplotypes. A phylogenetic tree was inferred using Molecular Evolutionary Genetic Analyses version 2.1. On the basis of bootstrap values from the phylogenic and molecular evolutionary analyses (20,21), haplotypes were sorted into three major branches of related haplotype groups, defined as clades. These three haplotype clades can be distinguished by the two polymorphisms −174G/C (rs1800795) and 1888G/T (rs1554606).
Genotyping was performed using TaqMan (Applied Biosystems, Foster City, CA) as the primary method. A length-modified single base extension protocol (22) was used when the TaqMan method failed, as was the case for the −174G/C polymorphism. The κ statistic, based on 45 blindly split samples from the CHOICE cohort, ranged from 93 to 100% for the four polymorphisms.
To assess for potential population stratification, a panel of 87 ancestry-informative single-nucleotide polymorphisms were genotyped to measure admixture. The degree of individual genetic white to black admixture was estimated using Bayesian methods implemented in the STRUCTURE program ver. 2 (23).
Biochemical Measurements
IL-6 was measured in serum by an ultrasensitive ELISA method (R&D Systems, Minneapolis, MN) with a coefficient of variation of 7%. Serum albumin levels were measured using the Bromocresol Green method (coefficient of variation 1.1%).
Outcome Ascertainment
CVD events were ascertained using follow-up through the dialysis clinics and Center for Medicare and Medicaid Services data. Incident CVD events included myocardial infarction, cerebrovascular accident, coronary artery bypass graft, percutaneous coronary angioplasty, peripheral artery bypass, amputation, abdominal aortic aneurysm repair, carotid endarterectomy, and sudden coronary death. Medical records from hospitalizations were reviewed and adjudicated by two members of the study’s outcomes committee using uniformly applied criteria modified from the Cardiovascular Health Study (24). The κ statistic for the event adjudication was 95%.
Statistical Analyses
Single-locus analyses were performed for the four polymorphisms. Dominant mode of inheritance, which was suggested in the previous studies (4), was tested here. Haplotype analyses were conducted for the two clade tagging polymorphisms −174G/C and 1888G/T. Individuals were assigned the most likely pair of haplotypes (when the probability of assignment was >90%) using the PHASE program. The distribution of IL-6 levels was highly skewed to the right, and logarithmic transformations were applied for normalization. All regression analyses were performed using STATA 7.0 statistical software (StataCorp, College Station, TX). Mean levels of inflammatory markers are presented after adjustment to avoid confounding. Adjustment was arbitrarily set to female; 60 yr; white; on hemodialysis; and no comorbidity, including diabetes, congestive heart failure, and prevalent CVD. Choosing other levels would change the absolute level but not the pattern of association. For survival analysis, follow-up time was defined as the period from initiation of dialysis to the first CVD event. Individuals were censored as a result of renal transplantation (n = 131), loss to follow-up (n = 3), or death attributed to causes other than CVD (n = 114). For all survival analyses, the proportionality assumption of the Cox model was confirmed by inspection of log (−log[survival function]) curves and Schoenfeld residuals.
Results
Table 1 shows the characteristics of 775 individuals according to race. Black patients tended to be younger and were more likely to be female, current smokers, and on hemodialysis. Black patients less frequently presented history of CVD and congestive heart failure. Black patients also had higher body mass index (BMI) and systolic BP and lower levels of IL-6. The genotype frequencies of the 11 IL6 polymorphisms in white patients were significantly different from those in black patients, and Hardy-Weinberg expectations were met in both races. The frequency of −174C allele (0.43 in white patients; 0.09 in black patients) was similar to that in the general population. The 32Ser allele was common in black patients and absent in white patients, whereas the 162Val allele was absent in black patients and rare in white patients. Clades 1 and 2 were more frequent, and clade 3 was less frequent in black than in white patients.
IL6 Polymorphisms, Levels of Inflammatory Markers, Incident CVD, and All-Cause Mortality
Compared with G/G homozygotes, carriers of the −174C allele had higher IL-6 levels and lower albumin (markers of inflammation) overall and in white patients, although the difference was marginally significant (Figure 2). This trend was not apparent in black patients.
Compared with 32Pro allele homozygotes, the Ser allele carriers had lower IL-6 levels and higher albumin levels. The 162Val allele, present on the −174C allele background, was significantly associated with lower IL-6 levels and higher albumin levels.
Over a median of 2.5 yr of follow-up, 294 CVD events occurred. Kaplan-Meier plot (Figure 3) and Cox proportional hazards model showed that compared with individuals with the genotype −174G/G, CVD risk was higher for GC heterozygotes and CC homozygotes and the hazard ratio (HR) of CVD was 1.81 (95% confidence interval [CI] 1.39 to 2.36) for GC and 1.37 (95% CI 1.00 to 1.89) for CC. A dominant inheritance model in which CC individuals were not at higher risk than GC individuals is consistent with previous studies (4). After adjustment for demographic information, diabetes, congestive heart failure, prevalent CVD, and comorbidity score (model a), these estimates diminished somewhat (HR 1.49; [95% CI 1.15 to 1.94] and HR 1.16 [95% CI 0.81 to 1.65], respectively). Both Pro32Ser and Asp162Val polymorphisms were associated with lower risk for CVD, but these associations were not significant. The 1888G/T polymorphism was not associated with levels of inflammatory markers or CVD risk.
IL6 Haplotypes, Levels of Inflammatory Markers, Incident CVD, and All-Cause Mortality
Haplotype analyses revealed patterns that were similar to the single-locus analyses (Table 2). Given that the 162Val allele had an opposite effect from the −174C allele and is present on the background of the −174C allele, the haplotype that contained the 162Val allele (n = 10) was removed from clade 3 (most of the −174C carriers). Clade 3 in the absence of 162Val was associated with higher levels of IL-6 (P = 0.03) and lower levels of albumin (P = 0.06). Clade 1 and clade 2 were not related with levels of inflammatory markers. Inclusion and exclusion of the haplotype that contained the 32Ser allele from clade 1 did not change the effect of clade 1.
In a dominant association model, clade 3 was associated with higher risk for CVD in overall and white patients, and clade 2 was associated with lower risk for CVD only in white patients. When compared with carriers of two copies of clade 1 (with 79 incident CVD events), clade 3 carriers (180 CVD events) but not clade 2 carriers (42 CVD events) were significantly associated with higher CVD risk. This suggests that the clade 2 effect was most likely due to the mirror effect of clade 3. With adjustment for covariates, the relative risk for CVD was 1.44 (95% CI 1.12 to 1.84; P = 0.006) for clade 3 carriers compared with clade 3 noncarriers (Table 2). This association remains significant after Bonferroni correction for three comparisons (at a significance level of 0.016). Further adjustment for systolic BP, BMI, cholesterol, HDL cholesterol, and IL-6 levels attenuated this association (HR 1.41; 95% CI 1.01 to 1.96). The pattern of increased CVD risk in individuals with clade 3 was predominantly seen in white but not in black patients. No significant interactions were detected with race, smoking, age (<60 versus ≥60 yr), gender, or diabetes for CVD or mortality. All regression models were rerun with adjustment of genetic admixture. No significant changes were observed with this adjustment.
We also conducted further analyses to identify an “at risk” haplotype in clade 3. Except for the two rare haplotypes (the first and fourth ones), the two common haplotypes in clade 3 were similarly associated with higher CVD risk (the second one: HR 1.45, P = 0.06; the third one: HR 1.67, P = 0.002). Other additional haplotype analyses did not increase the predictive value of IL6 variants for inflammatory markers and CVD risk.
Discussion
In this large, prospective study of dialysis patients, we first reported that two coding variants in the IL6 gene, pro32Ser and Asp162Val, seem to downregulate the inflammatory process by lowering IL-6 and elevating serum albumin levels. The two variants are not significantly associated with reduced CVD risk, which could be due to a limited power related to the rarity of the two variants. Information on their functional significance is lacking. Our findings suggest that these coding variants may alter IL-6’s structural stability or function at the protein level, because amino acid substitutions from aspartic acid to valine and from proline to serine can have important structural and functional consequences (25).
The data confirmed that the polymorphism −174G/C predicted incident CVD and mortality in white patients. The modest dominant effect of the −174C allele that was observed in the dialysis population is consistent with that in the general population reported by two large-scale prospective studies (4,7) and two case-control studies (5,6) but not with other case-control studies (8,9,26,27). The previous difficulty demonstrating a significant association highlights two common problems. One is survival bias (e.g., early death attributable to genotype), which occurs particularly in a cross-sectional or case-control study. The other one is inadequate sample size to detect true associations, which also may be the case for our black subgroup analysis given the lower frequency of the −174C allele in this group. The consistency of prospective data in diverse cohorts of white patients may mitigate the concern of confounding as a result of population stratification.
Our data revealed that the −174C allele in the absence of the 162Val variant (clade 3) predicted higher serum levels of IL-6 and lower albumin levels. Because the −174 polymorphism is close to a glucocorticoid receptor binding site that has a negative regulatory effect, a mutation to the C allele from the ancestral G allele might influence binding at this receptor (11,28) and lead to a decreased ability to repress transcriptional activation and result in overexpression of the IL6 gene during an inflammatory state. This hypothesis is supported by large in vivo studies in patients who had aneurysmal disease (7) or hypertension (29) or were postoperative (10) or in newborns after birth trauma (30). Those studies and ours shared a common setting where participants were exposed to inflammatory stimuli. Mixed results have been reported in healthy individuals (8,26,31–34) and in in vitro studies (11,12) of situations in which there is little or no inflammation and glucocorticoid regulation is not critical or absent. Nevertheless, inadequate sample sizes, confounding, gene–environment interactions, allelic (e.g., the −174C effect may differ in the presence or absence of the 162Val) or locus heterogeneity, or a nonfunctional variant under study also may explain this inconsistency.
Our results indicate that functional variations in the IL6 gene may modify CVD risk by influencing serum IL-6 levels and in some cases changing the structure of the IL-6 protein. These findings support an atherogenic role of IL-6 because genotypes precede atherosclerosis and do not change over time. The acute-phase reaction, trigged by upstream cytokines such as IL-6, likely are involved in atherosclerosis through endothelial activation, adhesion molecules release (35), vascular smooth muscle cell proliferation (36), platelet aggregability, and/or coagulation (37).
Our study has several limitations. Inflammatory markers were measured on only one occasion; multiple measurements would provide a more precise estimate of the true values. Unmeasured variability of circulating IL-6 levels within individuals may partially explain the observed residual effect of IL6 gene variants after adjustment for serum IL-6 levels. Given the long duration of the processes that leads up to ESRD, people with variant alleles that lead to higher IL-6 levels may have died of CVD before developing ESRD. Such selective mortality leads to an underestimation of the risk associated with variant alleles. In this study, we were unable to sequence a polymorphic tract of A and T residues (AnTn) at position −373, which was suggested to be potentially functional (12,38). Therefore, we cannot exclude the possibility that additional functional variants in or near IL6 explain these associations. In addition, null association was observed in black patients. We have limited power to examine racial difference, so the ability to generalize these findings to black patients is uncertain. The current genotyping study did not include all of the study participants because of missing genotype information. The baseline characteristics of patients who were not included in the genotyping study were similar to those who were included in terms of age, gender, race, smoking, BMI, BP, serum total cholesterol, history of diabetes, and CVD event rate (all comparisons P > 0.38).
Conclusion
Two missense variants (Pro32Ser and Asp162Val) and one variant (−174G/C) in the promoter of the IL6 gene influence trait levels. The −174G/C polymorphism is a strong and independent predictor of clinically evident CVD. This study was limited to dialysis patients who experience both a high level of inflammation and high risk for CVD and mortality and may have different pathophysiology of CVD from the general population. In white patients, because the −174C allele occurs at such high frequency (approximately 43% in the general population [4]), its public impact is considerable (HR of 1.44 corresponds to a population attributable CVD risk of approximately 25%). The internal consistency across several outcomes (measures of inflammation and CVD), together with the presence of high levels of IL6 mRNA in atherosclerotic arteries seen in other studies, provides evidence that high inflammatory responsiveness to environmental stimuli can determine CVD risk. Further evaluation of the three IL6 variants (pro32Ser, Asp162Val, and −174G/C) in both prospective studies and clinical trials should precede tailored pharmacologic therapy in both the high inflammation dialysis population and other populations. Our study supports the common disease-common variant hypothesis (39) and also highlights the importance of less frequent but biologically important variants in characterizing genes for complex diseases.
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Figure 1: Phylogenetic relationships of
IL6 gene haplotypes on chromosome 7p21. On the basis of resequencing data of 31 single-nucleotide polymorphisms (SNP) from 23 European and 24 African-American patients (
13), a phylogenic tree of the
IL6 gene that was constructed using Molecular Evolutionary Genetic Analyses reveals three major branches: Clades 1 to 3. The two polymorphisms highlighted in gray and marked with a star (−174 G/C and 1888G/T) were chosen as “clade tag” SNP. The numbers to the right are the frequencies (%) for black and white CHOICE participants of the corresponding haplotypes. Dots represent the same alleles as those on the ancestral haplotype on the bottom, and dashes denote deletions. The 11 genotyped SNP are underlined in the figure. The 31 SNP on the bottom line are as follows from left to right: rs2069824,
rs2069825,
rs2069827,
rs1800797,
rs1800796 (
−597G/A),
rs1800795 (
−572G/C),
rs2069830 (
−174G/C), rs2069832, rs2069833, rs1474348, rs2069838, rs1474347, rs1524107, rs2066992, rs2069833,
rs2069840,
rs1554606, rs2069841, rs2069842, rs1548216, rs2069843, rs2069844,
rs2069845, rs2069847, IL6#5602,
rs2069860,
rs2069849, rs2069852, rs2069855, Il6#7592, and Il6#7659. IL-6 numbers are from the Programs for Genomic Applications annotated sequence (
http://pga.gs.washington.edu/data/il6/il6.ColorFasta.html).
Mean levels of serum inflammatory markers associated with potential functional variants in the IL6 gene, adjusted to female; 60 yr; white; on hemodialysis; and no comorbidity, including diabetes, congestive heart failure, and prevalent cardiovascular disease (CVD). P values were presented for dominant model of inheritance.
Figure 3: Unadjusted cumulative incidence of CVD according to −174G/C genotype.
Table 1: Patient characteristics by race (n = 775)a
Table 2: Adjusteda difference in mean levels of inflammatory markers and adjusted HR of incident CVD associated with the clades in the IL6 gene
CHOICE is supported by RO1-HL-62985 (National Heart, Lung, and Blood Institute), RO1-DK-59616 (National Institute of Diabetes and Digestive and Kidney Diseases), R01-HS-08365 (Agency for Healthcare Research and Quality), and a grant from Baxter Healthcare Corporation. J.C. is supported in part as an American Heart Association established investigator (01-4019-7N). M.J.K. is supported by K24-DK-02856 (National Institute of Diabetes and Digestive and Kidney Diseases). R.P.T. is supported by HL 46696 and HL 58329. This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. This publication has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-12400.
We thank the patients, staff, laboratory, physicians who participated in the CHOICE Study at Dialysis Clinic, Inc., and Johns Hopkins University, and the Cardiovascular Endpoint Committee (current members: Bernard G. Jaar, MD, MPH, Yongmei Liu, MD, Joseph A. Eustace, MD, MHS, Richard M. Ugarte, MD, Melanie H. Katzman, MD, MHS, and J. Craig Longenecker, MD, PhD; former members: Michael Klag, MD, MPH, Neil R. Powe, MD, MPH, MBA, Michael J. Choi, MD, Renuka Sothinathan, MD, MHS, and Caroline Fox, MD, MPH). Cardiovascular events adjudicators are Nancy E. Fink, MPH, and Laura C. Plantiga, ScM.
Published online ahead of print. Publication date available at www.jasn.org.
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