The aim of the study was to investigate the relationship among environmental risk factors, the methylation level of AR candidate genes reported from polymorphism association studies and AR risk in a cohort of children aged 3-6 years in China. We found that among the 17 investigated genes, the DNA methylation level of ADAM33 was significantly lower in the AR group than controls and the difference was still significant after correcting for multiple testing. Furthermore, we showed that pet exposure was related to the higher risk for AR with respect to DNA methylation level in promoter region of ADAM33.
In our study, maternal allergic history was a strong risk factor for AR among a cohort of Chinese children. This result is consistent with that of a previous study involving a cohort of 6 year-old children , reporting that maternal allergic history was associated with higher risk for AR development (25). The biological mechanism proposed was that childhood allergy development was impaired by maternal allergic disease history through impairment of neonatal regulatory T-cells (12). Furthermore, plenty of contradictory associations exist as whether furred pet exposure (cats and dogs) may be a risk or a protective factor for the AR development (16, 26, 27). We also found that pet exposure was another risk factor for AR, which is consistent with a recent study from Finland showing that dog and cat exposure in early life could increase the risk of developing pet allergies (28). However, the cumulative evidence from several systematic reviews suggest that pet allergen exposure has not increased the risk for developing allergic disease (16, 29, 30). The discrepancies are likely due to the ubiquitous nature of pet allergens, while pet owners are more concerned about sanitation and many other hygiene-related reasons.
Genetic association studies have advanced our understanding of genetic risk factors for allergic diseases. In the latest GWAS of AR, 41 AR-related risk loci have been reported, including 20 loci that had not previously been related to the disease (2-5), however, none of them have been confirmed to be a hub gene in the development or persistence of allergic diseases. In this study, 17 candidate genes for association with AR were identified using Human Genome Epidemiology (HuGE) Navigator (21) and methylation levels of promoter regions were compared in PBMCs of AR cases and control individuals.
One CpG site in the promoter region of ACE was found as the only CpG site remained significant after correcting for multiple testing in our study. Although methylation level of this CpG site was not significantly associated with the total eosinophil count in the case group, however, methylation of ACE might still play an important role in the development of AR, considering transcription of the mRNA might be regulated by DNA methylation at one specific site in its promoter (31).
Disintegrin and metalloproteinase 33 (ADAM33), the first asthma-susceptible gene identified by positional cloning, was the only gene identified with significant methylation level differences between the groups at the CpG site level, amplicon level and gene level. Notably, ADAM33 has been extensively reported as a susceptibility gene in bronchial hyperresponsiveness, asthma and AR (21, 32-34). ADAM33 is expressed in the smooth muscle, myofibroblasts, and fibroblasts of asthmatic airways, thus the function of this protein might be involved in the airway remodeling (35). Various lines of evidence from previous human and animal studies have indicated that the expression level of ADAM33 was upregulated during acute or chronic lung inflammation (36). Even though the existence of this functional link between ADAM33 and allergic airway inflammation, its role in the pathophysiology of AR is still to be clarified.
The dramatic increase in the prevalence of allergic diseases during the past decades is more likely to be the result of changes in environmental factors, accompanied by epigenetic changes in the human genome. By using Adam33−/− knock out mouse, a recent report has shown substantial interaction between Adam33-mediated airway remodeling and sensitivity to allergen exposure, leading to allergic inflammation and bronchial hyperresponsiveness in early life (37). The present work is the first study to report the association between methylation level of ADAM33 and AR risk in terms of the interaction between pet-exposure and ADAM33 gene promoter methylation. The underlying disease mechanism of this effect remains unknown. However, this study suggests that it is important to examine not only the effect of early-life risk factors, but also their interaction with the DNA methylation level of candidate AR genes.
There are several limitations to our study. First, we used a relatively small sample size, hence there could be a possibility of overestimating the significance of the association of ADAM33 methylation with AR. Furthermore, the fact that all the children recruited from only one ENT clinic might introduce selection bias into the study. However, we speculate that the relationship between ADAM33 methylation and pet exposure is involved in AR onset. Second, there were several risk factors that could confound the interaction between pet exposure and the DNA methylation levels of ADAM33 in children with AR, including disinfection habits of pet owners, mode of delivery, etc. Furthermore, since RNA quality was not good enough for measuring expression level of ADAM33, further studies are needed to investigate the potential differential expression pattern of ADAM33 in AR. To overcome these limitations, a prospective cohort study with bigger sample size will be conducted in the future.