TRPV1, TRPV4, and TRPA1 cation channels are members of the vanilloid (TRPV) and ankyrin (TRPA) subfamily of transient receptor potential channels. These channels are expressed in different cells of the lung, including sensory neurons participating in airway reflex responses, bronchial smooth muscle, and epithelial and endothelial cells 12. TRPV1 channels are activated by capsaicin, heat, particulate matter, and various noxious chemicals, and are upregulated in airway nerves and airways smooth muscle of individuals with cough 1234. It has recently been shown that the TRPV1 Ile585Val single nucleotide polymorphism (SNP) results in a loss-of-channel function, and that this SNP is associated with a lower risk of cough and wheezing among children with asthma 5. TRPA1 acts as a receptor for a wide range of irritants and chemicals, including air pollutants and some of the principal components of cigarette smoke 67. Agonists of TRPV1 and TRPA1 channels can elicit a reproducible cough response in humans 89, and induce neurogenic inflammatory responses in experimental models 71011. TRPV1 and TRPA1 have been put forward as major targets for novel anti-tussive drugs 1213.

TRPV4 could play a role in the pathogenesis of airway disease through regulation of endothelial and epithelial permeability in the lungs, bronchial smooth muscle cell contractility, and mucociliary transport 14151617. TRPV4 dysfunction has already been associated to airway diseases: airway epithelial cells from cystic fibrosis patients show a defective regulation of TRPV4 1819, and SNPs in the TRPV4 gene have been found to be associated with chronic obstructive pulmonary disease (COPD) 20.

Because of the central role of TRP channels in cough response, we hypothesized that variants in genes encoding TRP channels may be associated with cough symptoms. Moreover, given the critical involvement of TRP channels in irritant sensing, TRP variants may be particularly relevant for irritant-induced cough 21. Exposures to irritants, for instance in cigarette smoke, or in exposures at work can lead to a spectrum of asthma-related symptoms, including cough 21. In recent years, studies on asthma and chronic cough symptoms in workers exposed to irritant cleaning products have raised interest in chronic or repeated exposures to relatively low levels of irritants 222324. However, little is known about the mechanisms implicated in irritant-induced airway disease.

We aimed to study association of SNPs in candidate genes TRPV1, TRPV4, and TRPA1 with cough symptoms. In addition, we explored whether TRP variants modulate associations between occupational exposures and cough symptoms, or between smoking and cough symptoms. The study was carried out in adults from the French Epidemiological study on the Genetics and Environment of Asthma (EGEA) and from the European Community Respiratory Health Survey (ECRHS).

Personal characteristics and the prevalence of cough symptoms of the study population are shown in Table 1. Any type of cough was reported by 60.0% of subjects with asthma, and 33.7% of subjects without asthma. Among subjects with asthma, 25.8% reported both nocturnal cough and usual cough, 22.0% reported nocturnal cough but no usual cough, and 12.2% reported usual cough but no nocturnal cough. For subjects without asthma, these prevalence rates were 8.3%, 18.0%, and 7.4%, respectively. Women were less often exposed to VGDF than men, and were more often never smokers (p < 0.01). Exposure to VGDF was not associated with asthma (OR 0.99 [0.81-1.21]), and this association did not differ between men and women.

The present study showed significant associations between TRPV1 SNPs and cough symptoms among subjects without asthma from two independent European studies. Although we carried out a pooled analysis to increase the power of detecting associations of SNPs with cough symptoms, we verified that the results were similar in the EGEA and ECRHS studies, and reached the 5% significance level for five SNPs in each study. Random-effects meta-analysis of these SNPs showed similar estimates of the summary ORs with those obtained from the pooled analysis and no evidence of heterogeneity across the different studies and countries involved for three of these SNPs. Haplotype-based association analysis confirmed the single SNP analyses, and global p-values for association between TRPV1 haplotypes and cough were highly significant (p < 5 × 10^-6).

The functional relevance of these TRPV1 SNPs, or variants not genotyped but in high LD with one of these SNPs, is so far unknown. TRPV1 variants could result in either activity or expression changes of TRPV1 channels in airway nerves or airways smooth muscle, influencing sensitivity for TRPV1-activating agents among carriers of such a variant. In a recent report, Cantero-Recasens et al.5 have shown that a isoleucine-to-valine mutation at position 585 of the TRPV1 protein results in a 20-30% loss of channel function, and that the corresponding TRPV1 I585V (rs8065080) SNP is associated with a significantly lower risk of wheeze and cough in children with asthma. The structural explanation for the altered activity of TRPV1 585 V is not known at present, and channel activity of other TRPV1 variants needs to be evaluated. In the present analysis, TRPV1 I585V was also associated with a lower risk of nocturnal cough in EGEA adults with asthma (OR 0.62 [0.40-0.96], p = 0.03), but the association was not statistically significant in the pooled analysis. We did not find evidence of an association between common TRPA1 or TRPV4 variants and cough. The lack of association between SNPs in TRPA1 and cough was especially surprising, since many ubiquitous environmental irritants have been shown to activate TRPA1 receptors to cause cough 1321. The significant associations of TRPV1 variants with cough imply that our study population was large enough to reveal moderately increased or decreased risk estimates, suggesting that the lack of association cannot be attributed to a lack of power in our study. Our study was the first to explore associations of TRPA1 variants and respiratory outcomes. We cannot exclude the possibility that other respiratory outcomes such as bronchial hyperresponsiveness or more specific (irritant-induced) cough phenotypes are associated with these variants. Moreover, other unidentified rare genetic variants in TRPA1 or TRPV4 may play a role.

TRPV1 and TRPA1 are prime candidate genes for gene-environment interactions with exposures to a wide range of irritants and chemicals that may be encountered in the workplace, but also in air pollution and in cigarette smoke. The present findings seem to support this hypothesis by suggesting that interactions between TRPV1 SNPs and occupational exposures and smoking may modify the risk of cough symptoms. However, given the large number of tests performed, our findings on gene-environment interaction should be taken cautiously. Our observation that TRPV1 variants interact with irritants that may be present in occupational exposures or cigarette smoke to increase the risk of cough symptoms needs replication in other epidemiological or functional studies.

Chronic cough is one of the most frequent reasons for consultation with a primary care or respiratory physician, and may cause important adverse psychosocial and physical effects on patients' quality of life 36. There is an unmet need for effective anti-tussive drugs for cough patients 37. TRPV1 is being pursued as one of the potential therapeutic targets, and TRPV1 antagonists are being developed 1337. The results of our genetic association study seem to support the hypothesis that modulation of TRPV1 channel activity may provide therapeutic benefit in cough. However, some TRPV1 antagonists cause significant side effects on body temperature, which has necessitated the withdrawal of these compounds from clinical trials 13. To date, both the scientific community and pharmaceutical industry are centered in the finding of a TRPV1 modulator to treat pain, without affecting temperature homeostasis. Potentially, the finding of such a modulator could be used as an antitussive agent.

Although cough is frequently associated with asthma 29, it has also been shown that in a general population, chronic cough may present as an independent symptom 38. Further, recent observations of chronic cough in subjects without obvious respiratory disease such as asthma, have suggested the existence of a distinct clinical entity, the cough hypersensitivity syndrome 3940. The mechanisms of idiopathic cough are unclear, but the enhanced cough reflex in patients may result from increased sensitivity of cough receptors such as TRPV1 39. In non-asthmatic chronic cough patients, increased expression of TRPV1 was shown 3. In our study, TRPV1 polymorphisms were associated with cough, but only in subjects without asthma. It is worth noting that associations with cough in those without asthma hold for those without airflow limitation, i.e. in subjects with cough independent of asthma or COPD. Thus, it could be hypothesized that genetic variation in TRPV1 increases the risk of an enhanced cough reflex among subjects without asthma. In asthmatics, the situation may be more complex, and the (modest) effect of genetic variation on TRPV1 channel activity may be obscured by the influence of other mechanims such as airway inflammation. For example, in asthma patients, chronic cough is associated with poor control of asthma and the use of inhaled corticosteroids 41. To further elucidate the difference between subjects with and without asthma, studies with more specific cough phenotypes would be useful. Future replication studies could assess whether TRPV1 polymorphisms may be associated with greater cough sensitivity to inhaled capsaicin in cough patients (with and without asthma). Such a study could also include TRPV1 gene expression analysis before and after capsaicin challenge.

Women have greater cough sensitivity to inhaled capsaicin than men, as was shown in cough patients and healthy volunteers 4243. The reason for the greater sensitivity among women is unknown, and it is unlikely that smaller airway size and sex hormones explain these differences 4243. In the present study, cough prevalence was also strongly associated with female sex, in particular among asthmatics, but the associations between TRPV1 SNPs and cough symptoms were not different for men and women.

We used occupational exposure to vapors, gases, dust, and/or fumes as a proxy of work-related irritant exposure. VGDF comprises a wide variety of exposures to occupational agents, including irritants that may induce cough by activation of TRP receptors. One can assume that misclassification of irritant exposures by VGDF is of a non-differential nature, resulting in estimated measures of association that are biased toward the null, and a negative impact on the power to observe gene-environment interactions between VGDF exposure and TRP variants. Asthma was unrelated to VGDF exposure, a result consistent with previous results 24. However, exposure to VGDF may modify the expression of asthma by increasing cough, which appears to be long-lasting since not all subjects were currently exposed. Long-term respiratory effects of occupational irritant exposures have been shown before, for example among women formerly employed in domestic cleaning 22.

TRPV1 SNPs were associated with nocturnal, usual, and chronic cough in subjects without asthma from two independent studies in eight European countries. Irritant exposures such as cigarette smoking and occupational exposures were associated with cough symptoms. Exploratory findings on gene-environment interaction suggested that these associations may be enhanced by TRPV1 SNPs.

CI: Confidence interval; COPD: Chronic obstructive pulmonary disease; ECRHS: European Community Respiratory Health Survey; EGEA: Epidemiological study on the Genetics and Environment of Asthma; JEM: Job-exposure matrix; LD: Linkage disequilibrium; OR: Odds ratio; SNP: Single nucleotide polymorphism; TRPA: Transient receptor potential ankyrin; TRPV: Transient receptor potential vanilloid; VGDF: Vapors: gases: dusts: and/or fumes.

The authors declare that they have no competing interests.

LAMS, JMA, MAV, and FK were involved in the conception, hypotheses delineation, and design of the study. All the above authors and also EB, JRG, NLM, HK, AEC, IP, DJ, RV, CJ, JH, IG and ML participated in the acquisition of the data or the analysis and interpretation of such information. LAMS, MK and FK wrote the article. All authors reviewed and commented on the paper and JMA, JRG, AEC, RV, JH, EB, FD and MAV had substantial involvement in its revision prior to submission. All authors read and approved the final manuscript.