Genes and pathways underlying susceptibility to impaired lung function in the context of environmental tobacco smoke exposure

Detrimental effects of environmental tobacco smoke (ETS) exposure on the level of lung function have been shown in a number of studies. [1‐3] Moreover, polymorphisms in several genes such as the glutathione S-transferases (GSTs), [4, 5] and ADAM33, [6] interact with ETS exposure, thereby negatively affecting the level and decline of lung function. In other words, ETS exposure has differential effects in subjects carrying mutant alleles compared to wild type alleles. Thus far, studies assessing interactions between genetic variants and ETS exposure were driven by a-priori knowledge of biological pathways, for example detoxification of noxious particles and gases by proteins such as the glutathione S-transferases.

In contrast, genome-wide interaction (GWI) studies are hypothesis-free and may yield novel loci in addition to those already known to be associated with impaired lung function directly (i.e. in genome-wide association studies without taking exposure into account). [7] Findings from such studies may provide novel insights in molecular pathways underlying disease pathogenesis, e.g. the development of chronic obstructive pulmonary disease (COPD) characterized by airflow obstruction. In this first genome-wide gene-by-ETS exposure study, we aimed to identify novel susceptibility loci, and additionally explored biological pathways potentially underlying this susceptibility to impaired forced expiratory volume in one second (FEV₁) in the context of ETS exposure during adulthood.

The current study is the first to explore SNP-by-ETS exposure interactions on the level of FEV₁ during adulthood in a hypothesis-free genome-wide manner. We extended our findings to pathway level analysis and showed that several pathways, i.e. the apoptosis, p38 MAPK and TNF pathways, may be underlying susceptibility to impaired FEV₁ in the context of ETS exposure.

The SNP with the most significant interaction in the identification cohort was located in the gene coding for KCNH1, also known as ether-à-go-go (EAG1). KCNH1 is a voltage-gated potassium channel that is highly expressed on mast cells and macrophages in germinal centers of reactive lymph nodes, [16] which may indicate its involvement in immune responses. Moreover, both mRNA and protein expression of KCNH1 were up-regulated during epithelial-to-mesenchymal transition (EMT) of human lung tumor cells induced by TGFβ1. [17] Increased expression of EMT markers has been observed in the airways of smokers, especially those with COPD. [18] Although one SNP in KCNH1 (rs7526579) had the same direction of interaction effect in all three cohorts, it did not reach genome-wide significance after meta-analysis and did not reach nominal significance in at least one of the verification cohorts. Findings, therefore, remain speculative.

Two SNPs-by-ETS interactions identified in the LifeLines study had nominally significant p-values in at least one of the verification cohorts, i.e. rs11950494 in SAPALDIA and rs2090789 in the Rotterdam Study. SNP rs11950494 is located in the gene actin beta-like 2 (ACTBL2) and rs2090789 is located in a predicted non-coding RNA LOC100128993. Both genes are expressed in lung tissue (genecards.​org), however, at current little is known about their biological function in general or relevance to lung function specifically.

In the current study, we used a large and well documented homogeneous cohort of a general population, i.e. the LifeLines study, to assess SNP-by-ETS exposure interactions. We used a liberal p-value threshold (p < 10⁻⁴) in the identification analysis and attempted to verify the SNP-by-ETS exposure interactions in two independent cohorts, the SAPALDIA and Rotterdam studies. Only 2 SNP-by-ETS exposure interactions were replicated with a nominal p-value and with the same direction of effect, which is less than expected based on chance only (i.e. 5% of 45 SNPs = 2.25). Moreover, none of the SNPs reached the Bonferroni-corrected threshold for genome-wide significance (p-value = 2.19*10⁻⁷). Therefore interpretation of the results remains difficult and the implications of the outcomes uncertain. The replication cohorts were relatively small, which may have limited the power to significantly replicate our findings. Another reason for not finding significant interaction effects may be the rather crude assessment of ETS exposure. In general, measuring ETS exposure during adulthood is difficult, especially when using self-reports. Thus far, no GWI studies on ETS exposure during adulthood have been published, suggesting that either no studies have been performed, or that publication bias exists due to null findings. There were slight differences in characteristics between the cohorts, i.e. enrichment with asthmatics in SAPALDIA (40% asthmatics) and the older mean age of subjects in the Rotterdam Study. However, sensitivity analysis in the identification cohort suggested that effects estimates for the two marginally replicated SNPs did not change when only non-asthmatics were included. Moreover, SNP-by-ETS interaction effects rather get more than less pronounced in older (≥50 years) compared to younger subjects (<50 years) (data not shown). Interestingly, a final sensitivity analysis showed that associations of these two SNP-by-ETS interactions with FEV₁, did only remain in subjects without airway obstruction (FEV₁/FVC ≥ 70%) (data not shown), suggesting that genetic susceptibility to effects of ETS is less important when already having airway obstruction.

In addition to single SNP analysis we performed a pathway analysis based on interaction p-values in the LifeLines study. Compared to single SNP analysis, pathway analysis may have increased power to detect genetic associations of the phenotype with a gene set/pathway. [19] Three pathways were significantly or suggestively enriched, i.e. the apoptosis, p38 MAPK and TNF pathways. Interestingly all three pathways may mutually interact and have been previously implicated in the pathogenesis of COPD, a disease caused by an abnormal inflammatory response to noxious particles and gases leading to airflow obstruction.

Apoptosis is a programmed form of cell death. Previous investigations within the SAPALDIA study have found suggestive evidence that genetic variation in the apoptosis pathway modifies the effect of pack years smoked on the decline of FEV₁. [20] An imbalance between apoptosis and proliferation of alveolar epithelial and endothelial cells has been observed in the lungs of patients with COPD. [21] Apoptosis is regulated by various pathways. One of the pathways is a response to extracellular signals by binding of members of the tumor necrosis family, such as TNF-alpha with death receptor TNF-receptor 1. [21] For example, cigarette smoke exposure was shown to increase TNF-alpha expression. [22] This interaction between the different pathways was also reflected by the substantial overlap in genes enriched in the TNF-alpha (Table 6) and apoptosis pathways (Table 4) in the pathway analysis. Another pro-apoptotic pathway responds to physical and chemical stressors via the release of cytochrome C by mitochondria. Subsequent formation of an apoptosome activates several caspases which eventually initiate apoptosis. Interestingly, we identified an intronic SNP in APAF1 that interacted with ETS exposure (p-value = 5.88*10⁻⁵) in the identification cohort LifeLines, the expressed protein of this gene is part of this apoptosome initiating apoptosis (Additional file 1: Tables S1 and Table 4). However, this SNP-by-ETS exposure interaction was not replicated in the SAPALDIA or Rotterdam study.

The TNF pathway was suggestively enriched in the pathway analysis. TNF-alpha is a cytokine playing an important role in inflammation through its activation of several downstream signaling cascades, amongst others the p38 MAPK pathway. Levels of TNF-alpha have been shown to be increased in sputum of COPD patients compared to both non-smoking and smoking controls, and in response to air pollution exposure. [23, 24] The second suggestively enriched pathway was the p38 mitogen activated protein kinase (MAPK) pathway, this pathway has also been implicated in the development and/or maintenance of a number of chronic airway inflammatory diseases such as COPD. [25] The p38 MAPK pathway is activated by various environmental stressors, growth factors and cytokines and in turn regulates the expression of inflammatory cytokines such as TNF-alpha and may initiate apoptosis. [26] Increased activation of p38 MAPK was seen in alveolar walls and alveolar macrophages of COPD patients compared to non-smoking and smoking controls. [27].

The current study is the first to explore gene-by-ETS interactions related to the level of FEV₁ in a hypothesis-free genome-wide manner. Findings from the single SNP analysis remain uncertain, yet our pathway analysis suggests that pathways previously implicated in COPD pathogenesis may, as well, underlie susceptibility to impaired lung function in the context of ETS exposure.