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DNA methylation profiling in peripheral lung tissues of smokers and patients with COPD

View Article: PubMed Central - PubMed

ABSTRACT

Background: Epigenetics changes have been shown to be affected by cigarette smoking. Cigarette smoke (CS)-mediated DNA methylation can potentially affect several cellular and pathophysiological processes, acute exacerbations, and comorbidity in the lungs of patients with chronic obstructive pulmonary disease (COPD). We sought to determine whether genome-wide lung DNA methylation profiles of smokers and patients with COPD were significantly different from non-smokers. We isolated DNA from parenchymal lung tissues of patients including eight lifelong non-smokers, eight current smokers, and eight patients with COPD and analyzed the samples using Illumina’s Infinium HumanMethylation450 BeadChip.

Results: Our data revealed that the differentially methylated genes were related to top canonical pathways (e.g., G beta gamma signaling, mechanisms of cancer, and nNOS signaling in neurons), disease and disorders (organismal injury and abnormalities, cancer, and respiratory disease), and molecular and cellular functions (cell death and survival, cellular assembly and organization, cellular function and maintenance) in patients with COPD. The genome-wide DNA methylation analysis identified suggestive genes, such as NOS1AP, TNFAIP2, BID, GABRB1, ATXN7, and THOC7 with DNA methylation changes in COPD lung tissues that were further validated by pyrosequencing. Pyrosequencing validation confirmed hyper-methylation in smokers and patients with COPD as compared to non-smokers. However, we did not detect significant differences in DNA methylation for TNFAIP2, ATXN7, and THOC7 genes in smokers and COPD groups despite the changes observed in the genome-wide analysis.

Conclusions: Our study suggests that DNA methylation in suggestive genes, such as NOS1AP, BID, and GABRB1 may be used as epigenetic signatures in smokers and patients with COPD if the same is validated in a larger cohort. Future studies are required to correlate DNA methylation status with transcriptomics of selective genes identified in this study and elucidate their role and involvement in the progression of COPD and its exacerbations.

Electronic supplementary material: The online version of this article (doi:10.1186/s13148-017-0335-5) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Manhattan plots showing distribution of possible differentially methylated CpG sites identified in this study across chromosomes. a Differential methylation analysis between smokers and non-smokers presented by chromosomal location (x axis). b Differential methylation analysis between COPD and non-smokers presented by chromosomal location (x axis). c Differential methylation analysis between COPD and smokers presented by chromosomal location (x axis). The y axis represents the negative log P value of their association. The genes marked in blue color are the once validated by pyrosequencing analysis. The black dotted horizontal line indicates the genome-wide significance threshold of P < 0.001. The top candidates in each pairwise comparison were selected by test statistics and unadjusted P values for comparisons (P < 0.001 is commonly used as a cutoff for relatively small unadjusted raw P value)
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Fig1: Manhattan plots showing distribution of possible differentially methylated CpG sites identified in this study across chromosomes. a Differential methylation analysis between smokers and non-smokers presented by chromosomal location (x axis). b Differential methylation analysis between COPD and non-smokers presented by chromosomal location (x axis). c Differential methylation analysis between COPD and smokers presented by chromosomal location (x axis). The y axis represents the negative log P value of their association. The genes marked in blue color are the once validated by pyrosequencing analysis. The black dotted horizontal line indicates the genome-wide significance threshold of P < 0.001. The top candidates in each pairwise comparison were selected by test statistics and unadjusted P values for comparisons (P < 0.001 is commonly used as a cutoff for relatively small unadjusted raw P value)

Mentions: Manhattan plot was generated using the Manhattan.plot function in R. Manhattan plot showed the negative P values on log10 scale on each chromosome with relatively small unadjusted raw P values corresponding to larger–log10 (p value). The chromosome-wide distribution of CpG sites and their comparison groups (non-smokers vs. smokers, non-smokers vs. COPD, and COPD vs. smokers) were shown (Fig. 1a–c and Tables 2, 3, and 4). Similarly, volcano plot was generated using the volcano plot function in limma package in R. Volcano plot is a scatter plot of log odds ratios vs. log fold change. The top 10 CpGs sites and associated genes including others CpG sites that were chosen for validation by pyrosequencing (NOS1AP, TNFAIP2, BID, GABRB1, ATXN7, and THOC7) based on their biological function with corresponding DNA methylation loci that were differentially methylated between different comparison groups are shown in the volcano plots (Fig. 2a–c and Tables 2, 3, and 4). Venn diagram was generated using the Venn diagram function in limma package in R. Venn diagram shows the overlap of DNA methylation loci that are shared between different comparisons (Additional file 1: Figure S2). The total number of overlapping genes and probes under each comparison groups shown in Venn diagram were not the same since a few of these did not annotate to genes (Additional file 2: Table S1, Additional file 3: Table S2 and Additional file 4: Table S3). Heatmaps were generated using the heatmap.2 function in the gplot package in R. We have included the DNA methylation loci and samples groups (NS, non-smokers; S, smokers, and C, COPD) for cluster analysis using the hierarchical clustering method. The green color in the heatmap denotes hyper-methylated loci, and the red color in the heatmap denotes the hypo-methylated loci. The sample groups are denoted by different colors. The top 100 genes along with the genes chosen for validation by pyrosequencing were included as part of the cluster analysis in smoker vs. non-smokers (Fig. 3a), COPD vs. non-smokers (Fig. 3b), and COPD vs. smokers (Fig. 3c), including the list of DMPs (Additional file 11: Table S10, Additional file 12: Table S11 and Additional file 13: Table S12).Fig. 1


DNA methylation profiling in peripheral lung tissues of smokers and patients with COPD
Manhattan plots showing distribution of possible differentially methylated CpG sites identified in this study across chromosomes. a Differential methylation analysis between smokers and non-smokers presented by chromosomal location (x axis). b Differential methylation analysis between COPD and non-smokers presented by chromosomal location (x axis). c Differential methylation analysis between COPD and smokers presented by chromosomal location (x axis). The y axis represents the negative log P value of their association. The genes marked in blue color are the once validated by pyrosequencing analysis. The black dotted horizontal line indicates the genome-wide significance threshold of P < 0.001. The top candidates in each pairwise comparison were selected by test statistics and unadjusted P values for comparisons (P < 0.001 is commonly used as a cutoff for relatively small unadjusted raw P value)
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Related In: Results  -  Collection

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Fig1: Manhattan plots showing distribution of possible differentially methylated CpG sites identified in this study across chromosomes. a Differential methylation analysis between smokers and non-smokers presented by chromosomal location (x axis). b Differential methylation analysis between COPD and non-smokers presented by chromosomal location (x axis). c Differential methylation analysis between COPD and smokers presented by chromosomal location (x axis). The y axis represents the negative log P value of their association. The genes marked in blue color are the once validated by pyrosequencing analysis. The black dotted horizontal line indicates the genome-wide significance threshold of P < 0.001. The top candidates in each pairwise comparison were selected by test statistics and unadjusted P values for comparisons (P < 0.001 is commonly used as a cutoff for relatively small unadjusted raw P value)
Mentions: Manhattan plot was generated using the Manhattan.plot function in R. Manhattan plot showed the negative P values on log10 scale on each chromosome with relatively small unadjusted raw P values corresponding to larger–log10 (p value). The chromosome-wide distribution of CpG sites and their comparison groups (non-smokers vs. smokers, non-smokers vs. COPD, and COPD vs. smokers) were shown (Fig. 1a–c and Tables 2, 3, and 4). Similarly, volcano plot was generated using the volcano plot function in limma package in R. Volcano plot is a scatter plot of log odds ratios vs. log fold change. The top 10 CpGs sites and associated genes including others CpG sites that were chosen for validation by pyrosequencing (NOS1AP, TNFAIP2, BID, GABRB1, ATXN7, and THOC7) based on their biological function with corresponding DNA methylation loci that were differentially methylated between different comparison groups are shown in the volcano plots (Fig. 2a–c and Tables 2, 3, and 4). Venn diagram was generated using the Venn diagram function in limma package in R. Venn diagram shows the overlap of DNA methylation loci that are shared between different comparisons (Additional file 1: Figure S2). The total number of overlapping genes and probes under each comparison groups shown in Venn diagram were not the same since a few of these did not annotate to genes (Additional file 2: Table S1, Additional file 3: Table S2 and Additional file 4: Table S3). Heatmaps were generated using the heatmap.2 function in the gplot package in R. We have included the DNA methylation loci and samples groups (NS, non-smokers; S, smokers, and C, COPD) for cluster analysis using the hierarchical clustering method. The green color in the heatmap denotes hyper-methylated loci, and the red color in the heatmap denotes the hypo-methylated loci. The sample groups are denoted by different colors. The top 100 genes along with the genes chosen for validation by pyrosequencing were included as part of the cluster analysis in smoker vs. non-smokers (Fig. 3a), COPD vs. non-smokers (Fig. 3b), and COPD vs. smokers (Fig. 3c), including the list of DMPs (Additional file 11: Table S10, Additional file 12: Table S11 and Additional file 13: Table S12).Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Epigenetics changes have been shown to be affected by cigarette smoking. Cigarette smoke (CS)-mediated DNA methylation can potentially affect several cellular and pathophysiological processes, acute exacerbations, and comorbidity in the lungs of patients with chronic obstructive pulmonary disease (COPD). We sought to determine whether genome-wide lung DNA methylation profiles of smokers and patients with COPD were significantly different from non-smokers. We isolated DNA from parenchymal lung tissues of patients including eight lifelong non-smokers, eight current smokers, and eight patients with COPD and analyzed the samples using Illumina&rsquo;s Infinium HumanMethylation450 BeadChip.

Results: Our data revealed that the differentially methylated genes were related to top canonical pathways (e.g., G beta gamma signaling, mechanisms of cancer, and nNOS signaling in neurons), disease and disorders (organismal injury and abnormalities, cancer, and respiratory disease), and molecular and cellular functions (cell death and survival, cellular assembly and organization, cellular function and maintenance) in patients with COPD. The genome-wide DNA methylation analysis identified suggestive genes, such as NOS1AP, TNFAIP2, BID, GABRB1, ATXN7, and THOC7 with DNA methylation changes in COPD lung tissues that were further validated by pyrosequencing. Pyrosequencing validation confirmed hyper-methylation in smokers and patients with COPD as compared to non-smokers. However, we did not detect significant differences in DNA methylation for TNFAIP2, ATXN7, and THOC7 genes in smokers and COPD groups despite the changes observed in the genome-wide analysis.

Conclusions: Our study suggests that DNA&nbsp;methylation in suggestive genes, such as NOS1AP, BID, and GABRB1 may be used as epigenetic signatures in smokers and patients with COPD if the same is validated in a larger cohort. Future studies are required to correlate DNA methylation status with transcriptomics of selective genes identified in this study and elucidate their role and involvement in the progression of COPD and its exacerbations.

Electronic supplementary material: The online version of this article (doi:10.1186/s13148-017-0335-5) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus