Association Analysis Of Genome-wide DNA Methylation With Long-term Ambient Air Pollution Exposure

Ambient air pollution has become a major risk factor for human health,especially in developing countries,such as China.Epidemiological studies suggested that long-term exposure to ambient air pollution is correlated with multiple diseases.However,the mechanisms underlying these impacts are poorly understood.As a crucial epigenetic modification,DNA methylation plays a key role in regulating gene expression and is susceptible to many environmental factors.Therefore,it is worth exploring whether ambient air pollution can mediate the development of diseases by affecting DNA methylation.Based on above,we carried out the following studies.（1）Collection of samples for studying the epigenetic effects of long-term exposure to ambient air pollutionObjectives:We aimed to establish two areas with distinct air pollution levels in China and to validate the adverse health consequence in the local residents so that we can conduct a case-control design.Materials and Methods:We established a highly polluted region（HPR）and a low-polluted region（LPR）in the provinces of Shanxi and Zhejiang,respectively.Air pollution data from the years 2015 to 2017 were obtained for six major ambient air pollutants(PM_2.5,PM₁₀,SO₂,NO₂,CO,and O₃)to evaluate the pollution levels.In addition,we collected9,607 particiants from the two regions and conducted an epidemiologic study to analyze the association between ambient air pollution and diseases.Results:Except for O₃,HPR had significantly higher concentrations of other five ambient air pollutants(PM_2.5,PM₁₀,SO₂,NO₂,and CO)and the largest fold-change was observed for SO₂（6.6-fold difference）.Concentration correlations,dynamic patterns along with time and primary pollutant proportions of the six pollutants in the two regions were similar.We converted concentration data into pollution levels and found most days in LPR were defined as good or excellent and polluted days were less than 10%.In contrast,most days in HPR were lightly polluted or good and approximately 40%days in HPR were polluted.Epidemiological study indicated that the prevalence of respiratory diseases（OR=3.63,95%CI=3.01-4.36）,cardiovascular diseases（OR=2.13,95%CI=1.75-2.62）,and allergies（OR=3.40,95%CI=2.76-4.25）were significantly higher in HPR.Conclusions:We established an HPR and an LPR in China based on the local air quality.Further,we validated the adverse health outcome associated with ambient air pollution in these two samples.Together,we provided a basis for studying the biological mechanism of diseases caused by ambient air pollution.（2）Impact of ambient air pollution on DNA methylationObjectives:The main aims of this study were to investigate the association between long-term ambient air pollution exposure and genome-wide DNA methylation and to identify involved differentially methylated regions（DMRs）,genes and pathways.Materials and Methods:We measured blood DNA methylation by whole-genome bisulfite sequencing（WGBS）in subjects from aforementioned HPR（N=72）and LPR（N=48）.We performed DMR analysis between participants of the two regions to study the association between long-term ambient air pollution exposure and DNA methylation.Additionally,we quantified mitochondrial DNA（mt DNA）copy number and plasma cytokines related to pathway analysis.Results:Genome-wide methylation analysis revealed 371 DMRs in subjects from the two areas and these DMR regions were located primarily in gene regulatory elements such as promoters and enhancers.Gene enrichment analysis showed that DMR-related genes were significantly enriched in diseases related to pulmonary disorders and cancers and in biological processes related to mitochondrial assembly and cytokine production.We found that HPR participants showed a higher mitochondrial DNA（mt DNA）copy number.Of those identified DMRs,15 were significantly correlated with mt DNA copy number.Further,cytokine assay indicated that an increased plasma interleukin-5（IL-5）concentration was associated with greater air pollution.Finally,by integrating the results of each analysis,we found five genes were supported by multiple evidence and four of them interacted with each other in a protein-protein association network.Conclusions:Our findings indicate that exposure to long-term ambient air pollution can lead to alterations in DNA methylation whose functions are related to mitochondria and immune responses.