Online supplement

Associations of IL6 polymorphisms with lung function decline and COPD

Jian-Qing He1*, Marilyn G. Foreman2*, Karey Shumansky1, Xuekui Zhang1, Loubna Akhabir1, Don D Sin​1, S F Paul Man1, Dawn L. DeMeo2, 3,Augusto A. Litonjua2,3,Edwin K. Silverman2,3, John E Connett4, Nicholas R Anthonisen5, Robert A Wise6, Peter D Paré1, Andrew J Sandford1

METHODS

Study participants:

LHS participants: A total of 1488 subjects were selected from the ~ 4,800 LHS subjects for whom DNA and serum are available. The LHS, sponsored by the National Heart, Lung and Blood Institute was a clinical trial to test the effect of an anti-smoking intervention and bronchodilator treatment on the progression of COPD 1. The LHS recruited a total of 5887 smokers aged 35-60 who had spirometric evidence of mild-moderate lung function impairment from 10 North American medical centers.1 Subjects were followed up yearly during the first five-year period1 and a venipuncture was performed at the fifth annual visit. Non-Hispanic whites account for the majority of participants and therefore participants from other ethnic groups were excluded in this study. In the manuscript the text refers only to the non-Hispanic whites.

These subjects were selected to form nested case control studies of two extreme phenotypes. Based on the rate of decline of lung function during the initial 5 year follow up in the 3,216 continuous smokers, we selected the 266 and 293 participants who had the fastest (rapid decline group) and slowest rate of decline of lung function (non-decline group), respectively. Arbitrary cut-off points of ≥ 3.0% predicted decrease /year in FEV1 and ≥ 0.4% predicted increase /year were used for rapid decliners and non-decliners, respectively. Based on the post bronchodilator FEV1% predicted at the start of the study, we selected 478 and 451 participants with the highest post bronchodilator FEV1% predicted (high baseline group) and the lowest post bronchodilator FEV1% predicted (low baseline group). Arbitrary cut-off points of FEV1 % predicted  88.9% and  67.0% were used for the high and low baseline groups, respectively. Since 130 participants from the rate of decline study group had baseline lung function within one of the categories for baseline lung function (54 individuals were in the high baseline range and 76 individuals were in the low baseline range), they were also analyzed in the baseline lung function study. Thus, there were 532 and 527 participants in the high and low baseline groups, respectively.

NETT-NAS Study participants: A subset of participants in the National Emphysema Treatment Trial (NETT)2,3 was analyzed in a nested case control study of IL6 polymorphisms and susceptibility to COPD. Briefly, the NETT was a randomized, multi-center, clinical trial comparing lung volume reduction surgery to conventional medical therapy in a cohort of individuals with severe COPD. We analyzed 389 non-Hispanic white subjects who were enrolled in the NETT Genetics Ancillary Study, a sub-study of the NETT. The control group was composed of 420 participants from the Normative Aging Study (NAS),4,5 a longitudinal study over the past four decades of healthy adult males that was initiated by the Boston Veterans Administration. The healthy smokers in the NAS cohort did not exhibit airflow obstruction and had 10-pack-year history of smoking at their last study visit.

TagSNP selection:

To select SNPs for genotyping in the LHS, the IL6 SNP discovery data were downloaded from the SeattleSNPs NHLBI Program for Genomic Applications, UW-FHCRC, Seattle, WA (URL: [accessed May 2003]. From all SNPs identified in the 23 unrelated European-American samples of the Centre d'Etude Polymorphisme Humain (CEPH) family panel, a set of tagSNPs was chosen for each gene using the LDSelect program developed by Carlson et al.6 This program selects a set of maximally informative tagSNPs based on their having linkage disequilibrium (LD) above a threshold with the non-selected SNPs. A relatively stringent LD threshold of r2>0.8 and minor allele frequency cutoff of 10% was used. Six SNPs in the IL6 gene (see Table E1) were selected. An assay for one SNP (rs2069840) in IL6 could not be established by the TaqMan method (Applied Biosystems, Foster City, CA). Therefore, five SNPs were genotyped in the LHS participants.An additional 2 SNPs were genotyped in the decline of lung function study by the Illumina GoldenGate method (Illumina Inc., San Diego, CA) i.e.rs2069840 that couldnot be genotyped by TaqMan, and rs1554606,selected for the NETT-NAS study.

In the NETT-NAS, six tag SNPs were selected with the ldSNP Tool, a linkage disequilibrium tagging algorithm, from the Innate Immunity in Heart, Lung and Blood Diseases PGA website ( SNP selection was limited to polymorphisms with a minor allele frequency of 0.1. SNPs not included in the panel were captured by an included SNP with an r2 0.8. Three additional IL6 SNPs were also genotyped in the panel.

Genotyping:

In the LHS, genotyping of the five selected tagSNPs was performed in 384 well plates in a total volume of 5 μl by the TaqMan 5’ exonuclease assay using primers and probes supplied by Applied Biosystems. Major and minor allele probes were labeled with 5’ FAM or 5’ VIC fluorophore as reporters (Applied Biosystems). The detection of the probe fluorescence signal was performed by an ABI PRISM 7900 Sequence Detection System (Applied Biosystems). Twelve DNA samples with known genotypes from the CEPH panel were included as positive controls and 8 no template wells were included as negative controls in each plate. No discrepancies were detected in the randomly selected 10% of the samples that were genotyped in duplicate. The additional 2 SNPs were genotyped using the Illumina Bead Array System in Génome Québec Innovation Centre.

In the NETT-NAS, the SNPs were genotyped on an Illumina BeadStation 500G System utilizing the GoldenGate assay technology (Illumina Golden Gate Assay, San Diego, CA). Genomic DNA was hybridized with a pool of assay oligonucleotides. Each assay consists of two allele-specific oligonucleotides and one locus-specific oligonucleotide. Allele-specific extension and ligation was performed, followed by PCR of the extended and ligated templates utilizing three universal products. The fluorescently labeled strands were hybridized to a Sentrix Array Matrix (SAM). The SAM is a fiberoptic assembly of arrays, each holding 1536 different oligonucleotide probe sequences, attached to uniquely detectable 3-micron beads that are assembled into microwells at the ends of optical fibers. The SAM was imaged with a BeadArray Reader, a two-channel, 0.8-micron resolution confocal laser scanner. Cy3 and Cy5 intensity values were collected for each sample array and normalized. The normalized intensities were converted to genotypes by GenCall software using a clustering algorithm to define and call genotypes.

Statistical Analysis:

Multiple logistic regressions for the two dichotomous outcomes, rate of decline and baseline lung function, were performed to test for the association with IL6 SNPs. These models were tested separately using the data sets corresponding to the outcome variables: rate of decline in lung function and baseline lung function. Confounding factors were evaluated and all models in which SNPs and IL 6 expression levels were compared with rate of decline were adjusted for age, gender, pack-years of smoking and research centre. All models in which SNPs and expression levels were compared with baseline lung function were adjusted for age, gender, pack-years of smoking, research centre, and FEV1/yr (% predicted post).

Haplotype association was tested using Hapassoc, a contributed R package available at This software performs likelihood inference of trait associations with haplotypes and other covariates for generalized linear models, including linear and logistic regression. Haplotype phase was not inferred and was handled with an expectation-maximization implementation of linear or logistic regression. An additive effect of haplotype on disease was assumed. Haplotypes of five loci in IL6 were considered for potential haplotype associations. All covariates previously considered in the single SNP association linear regression models were included in the linear regression models testing for association of haplotypes and expression levels. Similarly, covariates considered in the single SNP association logistic regression models were included in the logistic regression models testing for haplotype association with the lung function phenotypes of interest.

In the LHS, multiple linear regression was also used to test association of IL6 SNPs with IL6 expression level. Since IL6 expression levels were right skewed and residuals resulting from the multiple linear regressions were also right skewed, a log transformation was applied before using it as an outcome, making outcome variables and residuals relatively normal. The log-transformed IL6 expression variable had a tail at the right of the distribution, so four extreme outliers were trimmed from the upper end of the distribution making the distribution more closely fit a normal distribution.Analyses with and without the 4 outliers were conducted and the results were unchanged by the exclusion.

Confounding factors used in previous analyses and previously published articles were evaluated as potential confounding factors in the present analysis. These factors included body mass index (BMI), age, gender, pack-years of smoking, and smoking status (continuing smokers, intermittent smokers, and sustained quitters).

In the NETT-NAS, testing for Hardy-Weinberg equilibrium, determining allele and genotype frequencies, and performing genetic association analysis with Armitage trend tests was accomplished with SAS Genetics (Cary, NC). Odds ratios for susceptibility to COPD were determined from multivariable logistic regression models with additive genetic coding performed with SAS.

RESULTS:

Performance of tagSNPs:

Haplotypes from all SNPs with minor allele frequency ≥ 10% in 23 CEPH samples were estimated with an expectation-maximization algorithm using the R haplo.stats package. The performance of tagSNPs was investigated by comparing the number of actual haplotypes and number of effective haplotypes (ne) resolved with tagSNPs as compared with that inferred using all SNPs with minor allele frequency ≥ 10% in both the IL6 and CRP genes 6. As shown in Figure E1, 70.0% of actual haplotypes and 84.0% of effective haplotypes from all 12 SNPs with a minor allele frequency ≥ 10% were resolved by the five selected IL6 tagSNPs. Therefore, the tagSNPs selected in this study performed very well in capturing common haplotypes.

FIGURE LEGENDS:

Figure E1 Performance of five tagSNPs in theIL6 gene. The tagSNPs selected are denoted in red font and their alleles are shown in each box. The common allele is shown as a blue box and the rare allele as a yellow box. The selected tagSNPs resolve 70% (7 out of 10) of the actual haplotypes (haplotypes resolved are indicated by red shading) and 84.0% of the effective number (ne) of haplotypes derived from SNPs with minor allele frequency ≥ 10%.

Note:

1) Haplotypes were arranged by a phylogenetic tree inferred from MEGA 3.1 software. The bar of 0.05 represents phylogenetic distance, which is a measure of evolutionary divergence between two homologous sequences

2) ne was calculated as where pi is the frequency of the ith haplotype.

3) At position -1479, “I” or “D” denotes CT insertion or deletion.

4) Haplotype frequency was calculated using the expectation-maximization algorithm in Haplo.stats for R.

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Figure E1

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Table E1. TagSNP selection using the LDSelect program and the nomenclature for the SNPs.

Bin / SNP ID / SNP / Minor allele frequency / Position in Ref Sequence / Position in gene / Position in protein
1 / rs1800797 / G/A / A: 0.47 / 1086 / -598 (promoter) / -
1 / rs1800795* / G/C / C: 0.50 / 1510 / -174 (promoter) / -
1 / rs2069832 / A/G / G: 0.50 / 2298 / 615 (second intron) / -
1 / rs2069833 / C/T / T: 0.50 / 2529 / 846 (second intron) / -
1 / rs1474348 / C/G / G: 0.50 / 2773 / 1090 (second intron) / -
1 / rs1474347 / G/T / T: 0.50 / 2989 / 1306 (second intron)
2 / rs1554606 / T/G / G: 0.43 / 3572 / 1889 (third intron) / -
2 / rs2069845* / G/A / A: 0.45 / 5014 / 3331 (fourth intron) / -
3 / rs2069825* / CT/- / del: 0.46 / 205 / -1479 (promoter) / -
4 / rs2069840 / C/G / G: 0.37 / 3437 / 1754 (third intron) / -
5 / rs1818879* / G/A / A: 0.29 / 7592 / 5909 (3’ flanking region) / -
6 / rs2069827* / G/T / T: 0.11 / 321 / -1363 (promoter) / -

Note: Sites are ordered by linkage disequilibrium, with sites showing similar patterns of genotype put into the same bin. The position in the IL6 gene is numbered by denoting the first nucleotide of the transcript as +1 (position 1684 in the sequence AF372214). All SNPs with minor allele frequency >10% are tagSNPs. One SNP was genotyped in each bin and genotyped SNPs in two nested LHS studies are indicated by *.

TABLE E2: Associations of IL6 SNPs with circulating IL6 concentrations

Variation studied / Subjects studied / Association / Population / Sample size / Reference
Multiple tagSNPs / Elderly (Participants of Cardiovascular Health Study) / Yes, -174G/C, P = 0.04
1889G/T, P = 0.03
No / European American
African American / 4714
862 / 7
8 SNPs including
-572G/C and
-598C/T (in LD with -174G/C, individuals with -598T always have -174C) / Patients with ACS* / Borderline association for -572G/C, P = 0.07 (the CG genotype was associated with higher IL6 levels compared with the GG genotype);
No association for -598C/T, P = 0.95 / Swedish / 2704 / 8
ACS patients with subsequent cardiovascular events / Yes, -572G/C: P = 0.01 (the CG genotype had higher IL6 levels compared with the GG genotype)
No association for other SNPs including -598C/T. / Swedish / 369
Healthy controls / No / Swedish / 447
13 SNPs including
-174G/C / Elderly women (>70 years old) / No / Caucasian / 363 / 9
Yes, heterozygotes associated with high IL6 levels compared with -174GG homozygotes / African American / 100
Yes, homozygous -174GG with low IL6
P < 0.05 / Chianti population
(Caucasian) / 266
-174G/C / Elderly with pre-existing major disease** / Yes, P = 0.027 (frequency of -174C allele carriers increased from the second IL6 quartile upward with respect to the bottom quartile) / Italian / 388 / 10
Elderly without pre-existing major disease / No / Italian / 436
-174G/C / Diabetics with peripheral artery disease / Yes, P < 0.0001, the GG genotype associated with higher plasma IL6 levels compared with -174 C allele carriers / Italian / 146 / 11
Diabetics without peripheral artery disease / Yes, P < 0.0001, the GG genotype associated with higher plasma IL6 levels compared to -174 C allele carriers / Italian / 144
-174G/C / Myocardial infarction / Yes, P = 0.016, -174C allele carriers have higher IL6 levels compared with the GG genotype / Italian / 200 / 12
-174G/C / > 55 years old population / No / Dutch / 641 / 13
7 SNPs including
-174G/C / Healthy women / No / US whites (96%) / 737 / 14
-174G/C and
-572G/C / Myocardial infarction / No / Swedish / 208 / 15

Note: *ACS = acute coronary syndrome

**Pre-existing major diseases were any one of: myocardial infarction, congestive heart failure, peripheral vascular disease, cerebrovascular disease, dementia, chronic pulmonary disease, connective tissue disease, peptic ulcer disease, liver and kidney disease, diabetes, cancer.

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