IL-6 Haplotypes, Inflammation, and Risk for Cardiovascular Disease in a Multiethnic Dialysis Cohort
Yongmei Liu*,
Yvette Berthier-Schaad,,
Margaret D. Fallin,
Nancy E. Fink,
Russell P. Tracy,
Michael J. Klag,
Michael W. Smith,|| and
Josef Coresh
* Wake Forest University School of Medicine, Winston-Salem, North Carolina; Johns Hopkins Medical Institutions, Baltimore, Maryland; Laboratory of Genomic Diversity; || Basic Research Program, SAIC-Frederick, National Cancer Institute, Frederick, Maryland; and University of Vermont, Burlington, Vermont
Address correspondence to: Dr. Josef Coresh, Johns Hopkins University, 2024 East Monument Street, Suite 2-600, Baltimore, MD 21205. Phone: 410-955-0495; Fax: 410-955-0476; E-mail: coresh@jhu.edu
Received for publication May 5, 2005.
Accepted for publication December 29, 2005.
It is unknown whether IL-6, a central regulator of inflammation,is a cause of or just a marker of atherosclerosis. Studies ofgenetic susceptibility to inflammation, however, avoid the potentialfor reverse causality. Variation in IL6 gene was studied asa predictor of cardiovascular disease (CVD) risk in a cohortof 775 incident dialysis patients, in whom IL-6 levels are elevated.On the basis of published resequencing data on the IL6 gene,a phylogenetic tree with three main branches (clades 1 to 3)was constructed. Two "clade tag" polymorphisms, –174G/Cand 1888G/T, and two missense variants, Pro32Ser and Asp162Val,were genotyped. Circulating IL-6 and albumin were measured amedian of 5 mo after the start of dialysis. CVD events wereascertained from medical records. During a median follow-upof 2.5 yr, 294 CVD events occurred. The two coding variants,Pro32Ser (present only in black patients, 10% Ser allele) andAsp162Val (present only in white patients, 1% Val), were associatedwith lower levels of IL-6 and higher levels of albumin. Thecommon variant in the promoter region, –174G/C, was stronglyassociated with higher CVD risk and weakly with IL-6 levels.Clade 3 (–174C carriers in the absence of 162 Val allele)was associated with higher IL-6 levels (P = 0.03) and higherCVD risk (hazard ratio 1.44, P = 0.006) after adjustment forcovariates. The IL6 gene has functional variants that affectinflammation and risk for CVD among dialysis patients, supportinga causal role for IL6 in CVD.
Inflammation is strongly and independently associated with cardiovasculardisease (CVD) risk. It may explain the excess CVD risk in dialysispatients, in whom inflammation is common (1). Interleukin-6(IL-6) is a major proinflammatory cytokine that is central tothe inflammatory response, regulating the hepatic synthesisof acute-phase proteins, such as fibrinogen, C-reactive protein,and albumin. IL-6 mRNA is present in atherosclerotic arteriesat a 10- to 40-fold higher level than in nonatheroscleroticvessels, and elevated levels of IL-6 are associated with increasedrisk for CVD, suggesting that IL6 has a role in the pathogenesisof atherosclerosis (2,3). Whether the relationship of IL-6 withCVD is causal, however, is difficult to prove because atherosclerosisalso may induce the synthesis of inflammatory markers.
Establishing a role of genetic variants in susceptibility toCVD would bolster the inference that high levels of the inflammatoryresponse to environmental stimuli can lead to CVD, because geneticvariants cannot be a consequence of CVD. More light has beenshed on a common G/C polymorphism at position –174 inthe promoter region of the IL6 gene. The –174C allelehas been associated with higher risk for CVD incidence and mortalityin some studies of white patients (4–7) but not in others(8,9). Circulating concentrations of IL-6 are thought to belargely regulated at the level of expression; however, the roleof 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 alongwith the very high rate of CVD in dialysis patients may allowthe determination of subtle effects of the IL6 gene. Also, allelicheterogeneity should be considered because individual haplotypesmay have differential effects (12). We investigated the influenceof potential functional variants and common haplotypes in theIL6 gene on levels of gene expression, inflammatory marker,and CVD risk in a prospective study of dialysis patients.
Study Design and Population
The CHOICE study is a national prospective cohort study of 1041incident dialysis patients who are aged 19 to 95 yr and from81 dialysis clinics. The study was approved by the InstitutionalReview Boards, and participants provided written informed consent.The study design and enrollment criteria have been describedelsewhere (14). Participants were enrolled from October 1995to 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 participantsfor whom blood was drawn before a dialysis session at a medianof 5.0 mo from the initiation of dialysis (95% within 8.7 mo).Their baseline demographic and clinical data were obtained fromquestionnaires, as well as from hospital and clinic records.Prevalent CVD was defined as medical record documentation ofcoronary artery disease, cerebrovascular disease, or peripheralvascular disease. The level of cardiovascular and other comorbiditywas assessed by a trained nurse on the basis of medical recordsand clinic staff reports using the Index of Co-Existent Disease,a standardized and validated four-level scale that has beentested in multiple studies (15).
DNA Analysis
The resequencing data of 24 African-American and 23 Europeanpatients from the Program for Genomic Applications at the Universityof Washington (13) were used to choose sequence variants togenotype. On the basis of 31 retrieved variants in the IL6 gene,11 polymorphisms were selected in this study, including threepreviously 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). Haplotypeswere constructed separately for black and white patient usingthe PHASE program, version 1.0.1 (16).
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).
Because of a limited statistical power to detect effects ofthe multiple haplotypes and the multiple comparison issues,we sought a method to classify haplotypes. Cladistic method,in which the evolutionary history of the haplotype variationis estimated, has been proposed for phenotypic association study(17,18). Such an approach requires that the cladistic structurenot be disrupted by recombination and can be estimated. In theIL6 gene, most of the variants are in linkage disequilibriumand belong to a single haplotype block (defined using the ConfidenceInterval Method [19] implemented in Haploview 3.2). The cladisticapproach, therefore, was chosen for grouping haplotypes. A phylogenetictree was inferred using Molecular Evolutionary Genetic Analysesversion 2.1. On the basis of bootstrap values from the phylogenicand molecular evolutionary analyses (20,21), haplotypes weresorted into three major branches of related haplotype groups,defined as clades. These three haplotype clades can be distinguishedby the two polymorphisms –174G/C (rs1800795) and 1888G/T(rs1554606).
Genotyping was performed using TaqMan (Applied Biosystems, FosterCity, CA) as the primary method. A length-modified single baseextension protocol (22) was used when the TaqMan method failed,as was the case for the –174G/C polymorphism. The statistic,based on 45 blindly split samples from the CHOICE cohort, rangedfrom 93 to 100% for the four polymorphisms.
To assess for potential population stratification, a panel of87 ancestry-informative single-nucleotide polymorphisms weregenotyped to measure admixture. The degree of individual geneticwhite to black admixture was estimated using Bayesian methodsimplemented 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 variationof 7%. Serum albumin levels were measured using the BromocresolGreen method (coefficient of variation 1.1%).
Outcome Ascertainment
CVD events were ascertained using follow-up through the dialysisclinics and Center for Medicare and Medicaid Services data.Incident CVD events included myocardial infarction, cerebrovascularaccident, coronary artery bypass graft, percutaneous coronaryangioplasty, peripheral artery bypass, amputation, abdominalaortic aneurysm repair, carotid endarterectomy, and sudden coronarydeath. Medical records from hospitalizations were reviewed andadjudicated by two members of the study’s outcomes committeeusing uniformly applied criteria modified from the CardiovascularHealth Study (24). The statistic for the event adjudicationwas 95%.
Statistical Analyses
Single-locus analyses were performed for the four polymorphisms.Dominant mode of inheritance, which was suggested in the previousstudies (4), was tested here. Haplotype analyses were conductedfor 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 PHASEprogram. The distribution of IL-6 levels was highly skewed tothe right, and logarithmic transformations were applied fornormalization. All regression analyses were performed usingSTATA 7.0 statistical software (StataCorp, College Station,TX). Mean levels of inflammatory markers are presented afteradjustment to avoid confounding. Adjustment was arbitrarilyset to female; 60 yr; white; on hemodialysis; and no comorbidity,including diabetes, congestive heart failure, and prevalentCVD. Choosing other levels would change the absolute level butnot the pattern of association. For survival analysis, follow-uptime was defined as the period from initiation of dialysis tothe first CVD event. Individuals were censored as a result ofrenal transplantation (n = 131), loss to follow-up (n = 3),or death attributed to causes other than CVD (n = 114). Forall survival analyses, the proportionality assumption of theCox model was confirmed by inspection of log (–log[survivalfunction]) curves and Schoenfeld residuals.
Table 1 shows the characteristics of 775 individuals accordingto race. Black patients tended to be younger and were more likelyto be female, current smokers, and on hemodialysis. Black patientsless frequently presented history of CVD and congestive heartfailure. Black patients also had higher body mass index (BMI)and systolic BP and lower levels of IL-6. The genotype frequenciesof the 11 IL6 polymorphisms in white patients were significantlydifferent from those in black patients, and Hardy-Weinberg expectationswere met in both races. The frequency of –174C allele(0.43 in white patients; 0.09 in black patients) was similarto that in the general population. The 32Ser allele was commonin black patients and absent in white patients, whereas the162Val allele was absent in black patients and rare in whitepatients. Clades 1 and 2 were more frequent, and clade 3 wasless frequent in black than in white patients.
Table 1. Patient characteristics by race (n = 775)a
IL6 Polymorphisms, Levels of Inflammatory Markers, Incident CVD, and All-Cause Mortality
Compared with G/G homozygotes, carriers of the –174C allelehad higher IL-6 levels and lower albumin (markers of inflammation)overall and in white patients, although the difference was marginallysignificant (Figure 2). This trend was not apparent in blackpatients.
Figure 2. 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.
Compared with 32Pro allele homozygotes, the Ser allele carriershad lower IL-6 levels and higher albumin levels. The 162Valallele, present on the –174C allele background, was significantlyassociated 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 modelshowed that compared with individuals with the genotype –174G/G,CVD risk was higher for GC heterozygotes and CC homozygotesand 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) forCC. A dominant inheritance model in which CC individuals werenot at higher risk than GC individuals is consistent with previousstudies (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 withlower risk for CVD, but these associations were not significant.The 1888G/T polymorphism was not associated with levels of inflammatorymarkers or CVD risk.
Figure 3. Unadjusted cumulative incidence of CVD according to –174G/C genotype.
IL6 Haplotypes, Levels of Inflammatory Markers, Incident CVD, and All-Cause Mortality
Haplotype analyses revealed patterns that were similar to thesingle-locus analyses (Table 2). Given that the 162Val allelehad an opposite effect from the –174C allele and is presenton the background of the –174C allele, the haplotype thatcontained the 162Val allele (n = 10) was removed from clade3 (most of the –174C carriers). Clade 3 in the absenceof 162Val was associated with higher levels of IL-6 (P = 0.03)and lower levels of albumin (P = 0.06). Clade 1 and clade 2were not related with levels of inflammatory markers. Inclusionand exclusion of the haplotype that contained the 32Ser allelefrom clade 1 did not change the effect of clade 1.
Table 2. Adjusteda difference in mean levels of inflammatory markers and adjusted HR of incident CVD associated with the clades in the IL6 gene
In a dominant association model, clade 3 was associated withhigher risk for CVD in overall and white patients, and clade2 was associated with lower risk for CVD only in white patients.When compared with carriers of two copies of clade 1 (with 79incident CVD events), clade 3 carriers (180 CVD events) butnot clade 2 carriers (42 CVD events) were significantly associatedwith higher CVD risk. This suggests that the clade 2 effectwas most likely due to the mirror effect of clade 3. With adjustmentfor covariates, the relative risk for CVD was 1.44 (95% CI 1.12to 1.84; P = 0.006) for clade 3 carriers compared with clade3 noncarriers (Table 2). This association remains significantafter Bonferroni correction for three comparisons (at a significancelevel 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 CVDrisk in individuals with clade 3 was predominantly seen in whitebut not in black patients. No significant interactions weredetected with race, smoking, age (<60 versus60 yr), gender,or diabetes for CVD or mortality. All regression models werererun with adjustment of genetic admixture. No significant changeswere observed with this adjustment.
We also conducted further analyses to identify an "at risk"haplotype in clade 3. Except for the two rare haplotypes (thefirst and fourth ones), the two common haplotypes in clade 3were similarly associated with higher CVD risk (the second one:HR 1.45, P = 0.06; the third one: HR 1.67, P = 0.002). Otheradditional haplotype analyses did not increase the predictivevalue of IL6 variants for inflammatory markers and CVD risk.
In this large, prospective study of dialysis patients, we firstreported that two coding variants in the IL6 gene, pro32Serand Asp162Val, seem to downregulate the inflammatory processby lowering IL-6 and elevating serum albumin levels. The twovariants are not significantly associated with reduced CVD risk,which could be due to a limited power related to the rarityof the two variants. Information on their functional significanceis lacking. Our findings suggest that these coding variantsmay alter IL-6’s structural stability or function at theprotein level, because amino acid substitutions from asparticacid to valine and from proline to serine can have importantstructural and functional consequences (25).
The data confirmed that the polymorphism –174G/C predictedincident CVD and mortality in white patients. The modest dominanteffect of the –174C allele that was observed in the dialysispopulation is consistent with that in the general populationreported by two large-scale prospective studies (4,7) and twocase-control studies (5,6) but not with other case-control studies(8,9,26,27). The previous difficulty demonstrating a significantassociation highlights two common problems. One is survivalbias (e.g., early death attributable to genotype), which occursparticularly in a cross-sectional or case-control study. Theother one is inadequate sample size to detect true associations,which also may be the case for our black subgroup analysis giventhe lower frequency of the –174C allele in this group.The consistency of prospective data in diverse cohorts of whitepatients may mitigate the concern of confounding as a resultof population stratification.
Our data revealed that the –174C allele in the absenceof the 162Val variant (clade 3) predicted higher serum levelsof IL-6 and lower albumin levels. Because the –174 polymorphismis close to a glucocorticoid receptor binding site that hasa negative regulatory effect, a mutation to the C allele fromthe ancestral G allele might influence binding at this receptor(11,28) and lead to a decreased ability to repress transcriptionalactivation and result in overexpression of the IL6 gene duringan inflammatory state. This hypothesis is supported by largein vivo studies in patients who had aneurysmal disease (7) orhypertension (29) or were postoperative (10) or in newbornsafter birth trauma (30). Those studies and ours shared a commonsetting 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 thereis little or no inflammation and glucocorticoid regulation isnot critical or absent. Nevertheless, inadequate sample sizes,confounding, gene–environment interactions, allelic (e.g.,the –174C effect may differ in the presence or absenceof the 162Val) or locus heterogeneity, or a nonfunctional variantunder study also may explain this inconsistency.
Our results indicate that functional variations in the IL6 genemay modify CVD risk by influencing serum IL-6 levels and insome cases changing the structure of the IL-6 protein. Thesefindings support an atherogenic role of IL-6 because genotypesprecede atherosclerosis and do not change over time. The acute-phasereaction, trigged by upstream cytokines such as IL-6, likelyare involved in atherosclerosis through endothelial activation,adhesion molecules release (35), vascular smooth muscle cellproliferation (36), platelet aggregability, and/or coagulation(37).
Our study has several limitations. Inflammatory markers weremeasured on only one occasion; multiple measurements would providea more precise estimate of the true values. Unmeasured variabilityof circulating IL-6 levels within individuals may partiallyexplain the observed residual effect of IL6 gene variants afteradjustment for serum IL-6 levels. Given the long duration ofthe processes that leads up to ESRD, people with variant allelesthat lead to higher IL-6 levels may have died of CVD beforedeveloping ESRD. Such selective mortality leads to an underestimationof 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 potentiallyfunctional (12,38). Therefore, we cannot exclude the possibilitythat additional functional variants in or near IL6 explain theseassociations. In addition, null association was observed inblack patients. We have limited power to examine racial difference,so the ability to generalize these findings to black patientsis uncertain. The current genotyping study did not include allof the study participants because of missing genotype information.The baseline characteristics of patients who were not includedin the genotyping study were similar to those who were includedin terms of age, gender, race, smoking, BMI, BP, serum totalcholesterol, history of diabetes, and CVD event rate (all comparisonsP > 0.38).
Two missense variants (Pro32Ser and Asp162Val) and one variant(–174G/C) in the promoter of the IL6 gene influence traitlevels. The –174G/C polymorphism is a strong and independentpredictor of clinically evident CVD. This study was limitedto dialysis patients who experience both a high level of inflammationand high risk for CVD and mortality and may have different pathophysiologyof CVD from the general population. In white patients, becausethe –174C allele occurs at such high frequency (approximately43% in the general population [4]), its public impact is considerable(HR of 1.44 corresponds to a population attributable CVD riskof approximately 25%). The internal consistency across severaloutcomes (measures of inflammation and CVD), together with thepresence of high levels of IL6 mRNA in atherosclerotic arteriesseen in other studies, provides evidence that high inflammatoryresponsiveness to environmental stimuli can determine CVD risk.Further evaluation of the three IL6 variants (pro32Ser, Asp162Val,and –174G/C) in both prospective studies and clinicaltrials should precede tailored pharmacologic therapy in boththe 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 butbiologically important variants in characterizing genes forcomplex diseases.
Acknowledgments
CHOICE is supported by RO1-HL-62985 (National Heart, Lung, andBlood Institute), RO1-DK-59616 (National Institute of Diabetesand Digestive and Kidney Diseases), R01-HS-08365 (Agency forHealthcare Research and Quality), and a grant from Baxter HealthcareCorporation. J.C. is supported in part as an American HeartAssociation established investigator (01-4019-7N). M.J.K. issupported by K24-DK-02856 (National Institute of Diabetes andDigestive and Kidney Diseases). R.P.T. is supported by HL 46696and HL 58329. This research was supported by the IntramuralResearch Program of the National Institutes of Health, NationalCancer Institute, Center for Cancer Research. This publicationhas been funded in part with federal funds from the NationalCancer Institute, National Institutes of Health, under ContractNo. NO1-CO-12400.
We thank the patients, staff, laboratory, physicians who participatedin the CHOICE Study at Dialysis Clinic, Inc., and Johns HopkinsUniversity, and the Cardiovascular Endpoint Committee (currentmembers: 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.
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Abstract
Introduction
statistic, based on 45 blindly split samples from the CHOICE cohort, ranged from 93 to 100% for the four polymorphisms. 
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. 
