There are only three studies published on associations of IL6
SNPs with COPD. Seifart et al. reported that there was no association of IL6
_−174 with COPD.11
Broekhuizen et al. did not find an association between IL6
_−174 and a cachexia phenotype in COPD subjects.23
Recently, Córdoba-Lanús et al. reported that IL6
_−572 but not IL6
_−174 was associated with COPD.12
All three studies have relatively small sample sizes. The associations of IL6
SNPs with FEV1
decline in the current study are novel and are the most significant findings among all the studies we have published utilizing the LHS cohort.13–16,24–27
To strengthen our initial finding in the LHS, we incorporated an association study of IL6
SNPs with COPD in the NETT-NAS. All SNPs that were genotyped and in high LD with the IL6
_−174G/C showed significant or borderline association with rapid decline of lung function in the LHS and with COPD in the NETT-NAS. We believe that the strength of the associations, the concordant results with several SNPs in high LD with the IL6
_−174G/C SNP, the available previous functional data on IL6
_−174G/C, the replication in a second population and the biologic plausibility for association provide strong evidence that this is a true association.
We examined the association of IL6 SNPs with IL6 serum levels as well as relationships between IL6 serum levels and lung function decline. We did not find any associations. We also found that adjusting the associations between IL6 SNPs and lung function for serum CRP levels in the LHS had no effect on the strength of the associations (data not shown). Therefore, we did not find evidence that the associations we report were mediated through an influence on production of IL6 or CRP.
Studies that have examined the effects of IL6
SNPs on IL6 mRNA and protein expression have led to conflicting results. The first reporter gene study demonstrated that a construct containing the −174G allele had higher reporter gene expression in HeLa cells, both under basal conditions and after LPS or IL1 stimulation10
. However, a second reporter gene study showed that a construct containing −174C had higher IL1-induced expression in HeLa cells than that of the −174G construct, although the difference did not reach statistical significance.28
By comparison of the two different cell types, the authors concluded that there is a cell type-specific regulation of IL6 expression.28
Nine of the most recently published studies of IL6
SNPs with circulating IL6 concentrations are summarized in Table E2. A recent meta-analysis of 5659 subjects from seventeen studies concluded that the −174 IL6 SNP was not associated with circulating IL6 levels.29
There are several explanations for the lack of consistent associations. First, the IL6
_−174G/C polymorphism might not be a strong determinant of serum IL6 levels. Second, the serum half-life of IL6 is short. Serum IL6 levels show marked diurnal variability.30
The blood samples for IL6 measurement in most studies, including our own, were not taken at a specific time of the day. A third explanation is that the SNPs studied may not be the actual functional SNPs. Recently, Samuel and colleagues have identified a novel IL6
transcriptional regulatory region (−5307 to −5202) much farther from the transcription initiation site than IL6
This report coupled with more recent identification of a novel functional SNP, IL6
_−6331T/C (rs10499563), with the T allele preferentially binding to Oct-1 transcription factor and producing higher reporter gene expression, provides evidence that additional functional SNPs do exist in IL6
However, since IL6_−6331T/C is in low LD with IL6_−174, our finding is not likely to be explained by these new functional data.
SNPs are not related to IL6 levels then what is the basis for their association with FEV1
decline and COPD? One possible explanation is that the association is truly driven via local pulmonary IL6 expression or that it is driven by serum IL6 levels but that the variability and lability of serum IL6 levels obscures this relationship; FEV1
may reflect the average IL6 levels and thus the degree of lung inflammation over the years of the study. In addition, the SNPs could influence IL6 levels and thus lung inflammation during exacerbations but not the constitutive levels during stable periods. IL6 is a pleiotropic cytokine which also modulates expression of many other genes.33
It may be that it is the effect of the IL6
variants on these genes that is the underlying mechanism for the associations we observed.
How can we explain the observation that IL6
SNPs were not associated with baseline FEV1
in the LHS but were associated with the presence of COPD in the NETT-NAS study? The mean age of the LHS participants was 48 years as opposed to a mean age of 68 years for the NETT-NAS participants. Baseline FEV1
at age 48 is influenced both by maximal attained FEV1
at ~ 25 years of age and by the rate of decline of lung function after age 25.34
However, the relative contribution of rate of decline in lung function will be much greater by age 68 than at age 48. Thus, FEV1
at age 68 in the NETT-NAS participants is likely to largely reflect the rate of decline of lung function during their long smoking history whereas there is likely a weaker relationship of FEV1
decline and baseline lung function at age 48.
Compared with previous studies, strengths of this study include larger sample size and good power. This sample size has adequate power to detect common genetic risk variants as shown in our previous power analyses, for example, it has 80% power to detect a relative risk of 2.0 when the frequency of the risk factor is 10% or above.22
There are several potential limitations of this study. Firstly, population stratification could have led to false-positive results. However, it has been reported that significant false-positive associations are unlikely to arise from population stratification in the non-Hispanic white population, especially in well-designed, moderately-sized, case-control studies such as ours.35
In addition, there was no significant evidence of population stratification in the NETT-NAS cases and controls.17
Second, false positive results might have arisen from multiple comparisons. However the consistent results in the NETT-NAS replication study make false positive results unlikely. Third, we have not identified the causal SNP for the associations. The identification of a novel functional SNP IL6
_−6331T/C (rs10499563), which has low LD with IL6_−174G/C (rs1800795) with r2
of 0.169 in the CEU HapMap database, indicates that the control of IL6 transcription is likely to be complex.32
We cannot exclude the possibility that SNPs other than the IL6
_−174G/C are also causal SNPs. Finally, serum IL6 levels were measured at year 5 of the LHS, therefore it may not be appropriate to link IL6 levels at year 5 with the baseline FEV1
as well as the rate of decline of FEV1
during 5 year follow-up.
In summary, we report associations of IL6 variants with rate of decline of lung function and with smoking-induced COPD.