Association Of Ambient Black Carbon And DNA Methylation With Childhood Asthma In Beijing,China:A Panel Study

BackgroundAsthma is a complex and heterogeneous disease that affects childi-en’s health and quality of life for a long time.Air pollution such as particulate matter may be an important factor in the continued increase of Chinese children’s asthma in the past 30 years.Black carbon(BC)is the active core of the biological effects of fine particulate matter and one of the pollutants of traffic pollution.The stimulating effect of black carbon and its mediated epigenetic modifications(especially DNA methylation)on childhood asthma remains to be clarifiedObjective:A panel study was used to compare the differences in the effects of black carbon exposure on DNA methylation in children with asthma,and to explore the stimulating effects of black carbon and DNA methylation on children with asthmaMethods:For the study,40 children with asthma who were treated at the Beijing Capital Institute of Pediatrics from September 2016 to November 2017 and revisited at least once in different seasons and 40 non-asthmatic children(aged 6-14 years)living in the same urban area in Beijing were selected as the research subjects.A panel study was conducted to compare the association between atmospheric black carbon concentration and DNA methylation in asthmatic children in different seasons.For the exposure assessment of black carbon,TSI PM2.5(fine particulate matter)sampler was used to collect atmospheric black carbon from 32 representative sites in Beijing on the polytetrafluoroethylene(PTFE)filter membrane,and the black carbon content was analyzed using four-wavelength-optical reflectance measurement with using a lab-modified integrating sphere.The ordinary Kriging interpolation method was used to fit the black carbon exposure level of the research subjects.The DNA methylation levels of 160 sputum samples were detected using the Illumina 850K methylation chip Statistical analysis used R,using the P value corrected by Benjamini-Hochberg false discovery rate(FDR)as the threshold(FDR<0.05).The "ChAMP" package is used for quality control and standardization of DNA methylation data.The ImerTest package was used to fit a linear mixed-effect model to perform an epigenome-wide association analysis(EWAS)of black carbon exposure and wheezing in asthmatic children in different seasons.The clusterprofiler package in R was used to perform GO(Gene Ontology)enrichment analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway enrichment analysis.Results:1.The EWAS analysis of childhood asthma:After adjusting the age and gender,98986 differential methylation probes(DMPs)were found to be associated with wheezing in asthmatic children.These DMPs located in or near genes,including PIK3AP1,GPC6,LRRC3B.The three most significant pathways of KEGG enrichment analysis are axon guidance,ErbB signaling pathway and adhesion junction pathway2.The EWAS analysis of atopy:After adjusting for age and gender,153 CpG sites were found to be associated with atopy(P<0.001).These DMPs were annotated with 103 genes including NAGS,ROBO1 and CIB2.GO analysis and KEGG analysis found no statistically significant terms or pathways3.Analysis of asthma EWAS in different seasons:In winter,192454 DMPs and 115 differential methylation regions(DMRs)were found between the asthma group and the control group,and they could be annotated to genes such as TNXB,PKP3,FGFR2.In summer,5356 CpG sites were found to be differentially methylated between the asthma group and the control group(p<0.001)4.The EWAS analysis of black carbon exposure:After adjusting for ozone,505 CpG sites were found to be associated with black carbon exposure(p<0.001),and these CpG sites were annotated to 355 genes such as NCOA7,FOXK2 and FGD4.GO analysis of these genes revealed 2 molecular functional terms(protein serine/threonine kinase activity and guanyl-nucleotide exchange factor activity)and 1 biological process term(regulation of GTPase activity).No statistically significant pathway was found in KEGG enrichment analysis5.Association analysis of black carbon exposure and asthma-related CpG site methylation:After adjusting for ozone,temperature,and relative humidity,BC exposure was found to be significantly associated with 918(918 out of 98986)asthma-related CpG sites(p<0.05),which can be annotated to genes such as SDC3.GO enrichment analysis showed that the most significant terms were guanyl-nucleotide exchange factor activity,axon part and regulation of Ras protein signal transduction KEGG enrichment analysis only enriched the synthesis and secretion pathway of cortisol.However,after multiple adjustments,BC exposure was not significantly associated with asthma-related CpG sites(FDR>0.05).Conclusion:The sputum DNA methylation of asthmatic children is associated with asthma attacks;the axon guidance pathway may play an important role in the onset of childhood asthma BC exposure may regulate the trigger mechanism of childhood asthma by affecting the level of DNA methylation in genes related to cortisol synthesis and secretion pathways,axons or synaptic structures.