Discussion
In this study, we identified one CpG site associated with ClinR and a different CpG site associated with ComR from whole blood, that were replicated in independent cohorts. We also revealed that 25 CpG sites were associated with asthma remission phenotypes in nasal brushes. In the nasal dataset, two differentially methylated regions were previously observed in another DNA methylation study of asthma remission using bronchial biopsies. This is the first epigenome-wide association study of asthma remission in both whole blood and nasal epithelial cells. This study is important since understanding asthma remission may provide new leads for future asthma treatments, and epigenetic studies may help to reveal these cellular mechanisms.
The two replicated CpG sites were annotated to genes which were not known to be involved in asthma (remission) before. ClinR-associated CpG cg13378519 was located in the promoter region of PEX11B gene.PEX11B encodes a protein of the PEX11 family that participates in proliferation of peroxisomes21. Overexpression of thePEX11B gene in human cells induces peroxisome proliferation22,23. Our results could indicate a possible role of peroxisomal proliferation as being involved in asthma remission. ComR-associated CpG cg24788483 was located in theTCF7L2 gene and also correlated with the expression level of this gene. The TCF7L2 gene product is a transcription factor that plays a role in the Wnt signaling pathway. SNPs in TCF7L2 were previously reported to be strongly associated with type-2 diabetes and this gene plays a role in the pancreatic insulin secretory response to incretins24, 25. Previously, the risk of asthma was reported to be higher in type 2 diabetes patients26 and it has been suggested that treatment targeting insulin resistance may have a positive effect on asthma patients27. However, it has never been studied whether this treatment could contribute to asthma remission.
Although remission subjects do not have asthma symptoms any more, they still may be different from healthy people. Airway abnormalities such as basement membrane thickening still exist in clinical and complete remission subjects28. Vermeulen et al. reported that the DNA methylation profile in remission subjects is different from that in healthy subjects 10. Our data in whole blood for our significantly replicated CpG confirmed this observation. The methylation levels of the ClinR associated CpG (cg13378519) were different between ClinR and healthy subjects, which indicated that the methylation status of subjects with asthma remission may not simply return to that of healthy people.
It is yet unknown if mechanisms of remission include local, tissue specific airway (epithelial) effects, immune cell effects, or both. DNA methylation is tissue and cell-type specific. Matched data from whole blood and nasal brush samples enable us to compare DNA methylation profiles associated with asthma remission in two different tissues, as proxies of both immunological and airway specific mechanisms. Although we identified several interesting CpG sites related to remission in nasal epithelium, our results of nasal brushes still need further replication. In previous studies, several signals of DNA methylation were significantly associated with asthma in both whole blood and nasal epithelium9,29. In our study, no significant enriched cross-tissue effects of DNA methylation in asthma remission were identified, which we interpret that cells contributing to the differentially methylation patterns are not present in both compartments.
DNA methylation might be related to the regulation of gene expression, and eQTM analysis may help to understand the function of CpG sites. Among the eQTM genes that correlated with ClinR-associated CpG sites in whole blood, the protein encoded by PRKCH (Protein Kinase C Eta) plays a key role in epithelial tight junction regulation which is important in maintaining the integrity and function of the airway epithelial barrier30; PDE1B (Phosphodiesterase 1B) encoded a protein belonging to phosphodiesterases (PDEs) family, and various PDE inhibitors showed anti-inflammatory, anti-remodelling and bronchodilator effect and are potential treatment of asthma31. 99 eQTM genes in whole blood were enriched in eight pathways, among which three pathways were related to airway epithelial function: activation of SMO (Smoothen), cilium assembly and focal adhesion. The activation of SMO activity in bronchial epithelia enhanced the allergen-induced goblet cell metaplasia, which is defined as a reversible transformation of airway epithelial cells to mucous cells such as goblet cells and may occur in asthma32. Airway cilia are important for clearance of inhaled particles and pathogens. One study showed asthma patients had less ciliated cells in airway samples than healthy people33, and changes in ciliary function may be relevant for the development of asthma in children34. Focal adhesion between the cell membrane and matrix are essential elements of airway smooth muscle cells migration which may play an important role during the airway remodeling of persistent asthma35. Our results suggest that asthma remission associated CpG sites reflect a role for airway epithelial barrier and airway remodeling in asthma remission.
When comparing our results with those of Vermeulen et al10 in bronchial biopsies, one CpG (cg13525448) in their top list of remission vs persistent asthma reached nominal significance (P<0.05) in our data and showed the same direction in our results of ComR in whole blood (Table S13). This CpG site is annotated to LBX1 gene and TLX1 gene by position. Besides, among their DMRs associated with ClinR, regions at the genePTCHD3 and LOC100507389 also showed up in our results of ClinR in nasal brushes. SNPs in PTCHD3 gene were previously associated with asthma in African American children36.
There are strengths and limitations in this study. To the best of our knowledge, this is the only cohort worldwide to study asthma remission in nasal brushes, which enable us to compare the DNA methylation profile in relation to asthma remission in whole blood and nasal epithelium. However, because of the uniqueness of this data, we could not replicate our results in nasal brushes in another independent cohort. Besides, we had matched DNA methylation and gene expression data in nasal brushes, which help us to get a better understanding of the function of CpG sites associated with asthma remission in nasal brushes. Regarding the limitations of this study, firstly, this study was performed in a relatively small sample with limited number of ComR cases, and the results were replicated in EGEA which is also with few ComR cases, which lead to low power of replication of ComR results. Although we studied over 450,000 DNA methylation sites, this only represents 1.6 % of the human DNA methylome. Thirdly, we studied mixed contributions of cells in whole blood and nasal brushes; and although we corrected for cell types using established methods, potentially stronger results may be anticipated when studying pure cell types, as was previously shown for DNA methylation sites associated with asthma in purified blood eosinophils8. Finally, we used a cross sectional analysis at follow up to investigate asthma remission. Future studies, with prospective designs, should be performed to investigate if these DNA methylation sites can predict future asthma remission.
In conclusion, we identified replicable DNA methylation signals associated with clinical and complete asthma remission, which may play a role in peroxisome proliferation and Wnt signaling pathway. This could help in identifying the underlying mechanisms of asthma
remission and conversely also of the chronicity of the disease.