AbstractBackgroundThe causes of poor respiratory function and COPD are incompletely understood, but it is clear that genes and the environment play a role. As DNA methylation is under both genetic and environmental control, we hypothesised that investigation of differential methylation associated with these phenotypes would permit mechanistic insights, and improve prediction of COPD. We investigated genome-wide differential DNA methylation patterns using the recently released 850K Illumina EPIC array in the largest single population sample to date.MethodsEpigenome-wide association studies (EWASs) of respiratory function and COPD were performed in peripheral blood samples from the Generation Scotland: Scottish Family Health Study (GS:SFHS) cohort (N=3,791; 274 COPD cases and 2,928 controls). In independent COPD incidence data (N=150), significantly differentially methylated sites (DMSs; p<3.6×10−8) were evaluated for their added predictive power when added to a model including clinical variables, age, sex, height and smoking history using receiver operating characteristic analysis. The Lothian Birth Cohort 1936 (LBC1936) was used to replicate association (N=895) and prediction (N=178) results.FindingsWe identified 29 respiratory function and/or COPD associated DMSs, which mapped to genes involved in alternative splicing, JAK-STAT signalling, and axon guidance. In prediction analyses, we observed significant improvement in discrimination between COPD cases and controls (p<0.05) in independent GS:SFHS (p=0.014) and LBC1936 (p=0.018) datasets by adding DMSs to a clinical model.InterpretationIdentification of novel DMSs has provided insight into the molecular mechanisms regulating respiratory function and aided prediction of COPD risk.FundingWellcome Trust Strategic Award 10436/Z/14/Z.Research in contextEvidence before this studyWe searched for articles in PubMed published in English up to July 25, 2018, with the search terms “DNA methylation” and “respiratory function”, or “COPD”. We found some evidence for association between differential DNA methylation and both respiratory function and COPD. Of the twelve previous studies identified, eight used peripheral blood samples (sample size [N] range = 100-1,085) and four used lung tissue samples (N range = 24-160). The number of CpG loci analysed range from 27,578 to 485,512. These studies have not identified consistent changes in methylation, most likely due to a combination of factors including small sample sizes, technical issues, phenotypic definitions, and study design. In addition, no previous study has: analysed a sample from a large single cohort; used the recently released Illumina EPIC array (which assesses ~850,000 CpG loci); adjusted methylation data and phenotype for smoking history, or used both prevalent and incident COPD electronic health record data.Added value of this studyTo our knowledge, this is the largest single cohort epigenome-wide association study (EWAS) of respiratory function and COPD to date (N=3,791). After applying stringent genome-wide significance criteria (P <3.6×10−8), we found that DNA methylation levels at 29 CpG sites in peripheral blood were associated with respiratory function or COPD. Of these 29, seven were testable in an independent population sample: all seven showed consistent direction of effect between the two samples and three showed replication (p<0.007 [0.05/7 CpG sites tested]). Our results suggest that adjustment of both the phenotypic and the DNA methylation probe data for smoking history, which has not been carried out in previous studies, reduces the confounding effects of smoking, identifies larger numbers of associations, and reduces the heterogeneity of effects across smoking strata. We used gene set enrichment and pathway analyses, together with an approach that combines DNA methylation results with gene expression data to provide evidence for enrichment of differentially methylated sites in genes linked to alternative splicing, and JAK-STAT signalling and axon guidance. Finally, we demonstrated that the inclusion of DNA methylation data improves COPD risk prediction over established clinical variables alone in two independent datasets.Implications of all the available evidenceThere is now accumulating evidence that DNA methylation in peripheral blood is associated with respiratory function and COPD.Our study has shown that DNA methylation levels at 29 CpG sites are robustly associated with respiratory function and COPD, provide mechanistic insights, and can improve prediction of COPD risk. Further studies are warranted to improve understanding of the aetiology of COPD and to assess the utility of DNA methylation profiling in the clinical management of this condition.
A validation of Illumina EPIC array system with bisulfite-based amplicon sequencing
