DNA Methylation Is Predictive of Mortality in Current and Former Smokers

Abstract

Rationale: Smoking results in at least a decade lower life expectancy. Mortality among current smokers is two to three times as high as never smokers. DNA methylation is an epigenetic modification of the human genome that has been associated with both cigarette smoking and mortality.Objectives: We sought to identify DNA methylation marks in blood that are predictive of mortality in a subset of the COPDGene (Genetic Epidemiology of COPD) study, representing 101 deaths among 667 current and former smokers.Methods: We assayed genome-wide DNA methylation in non-Hispanic white smokers with and without chronic obstructive pulmonary disease (COPD) using blood samples from the COPDGene enrollment visit. We tested whether DNA methylation was associated with mortality in models adjusted for COPD status, age, sex, current smoking status, and pack-years of cigarette smoking. Replication was performed in a subset of 231 individuals from the ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) study.Measurements and Main Results: We identified seven CpG sites associated with mortality (false discovery rate < 20%) that replicated in the ECLIPSE cohort (P < 0.05). None of these marks were associated with longitudinal lung function decline in survivors, smoking history, or current smoking status. However, differential methylation of two replicated PIK3CD (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta) sites were associated with lung function at enrollment (P < 0.05). We also observed associations between DNA methylation and gene expression for the PIK3CD sites.Conclusions: This study is the first to identify variable DNA methylation associated with all-cause mortality in smokers with and without COPD. Evaluating predictive epigenomic marks of smokers in peripheral blood may allow for targeted risk stratification and aid in delivery of future tailored therapeutic interventions.

Keywords: DNA methylation; all-cause mortality; chronic obstructive pulmonary disease; epigenetics; survival analysis.

Figure 1.

Overview of the study illustrating the genome-wide analysis, intersection with previous findings, and replication of the results. COPD = chronic obstructive pulmonary disease; COPDGene = Genetic Epidemiology of COPD; DGE = differential gene expression; DNAm = DNA methylation; ECLIPSE = Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints; EWAS = epigenome-wide association study.

Figure 2.

Distribution of percent DNAm for the PIK3CD (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit δ) CpG sites in both COPDGene (Genetic Epidemiology of COPD) and ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints): (A) cg03971555 and (B) cg12033075. The distribution for both alive and deceased subjects is shown. COPD = chronic obstructive pulmonary disease; DNAm = DNA methylation.

Figure 3.

Survival curves for PIK3CD (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit δ) CpG sites in COPDGene (Genetic Epidemiology of COPD). (A) For the site cg03971555, the subjects alive at enrollment for each bin: n = 160 (red), n = 159 (green), n = 159 (blue), and n = 159 (black). Thirty samples have missing DNAm data (total n = 637). The subjects alive at last time point: n = 121 (red), n = 136 (green), n = 143 (blue), and n = 144 (black). (B) For the site cg12033075, the subjects alive at enrollment for each bin: n = 167 (red), n = 167 (green), n = 166 (blue), and n = 167 (black). The subjects alive at last time point: n = 124 (red), n = 144 (green), n = 148 (blue), and n = 150 (black). Marks show times with data censoring. The time unit is days. COPD = chronic obstructive pulmonary disease; DNAm = DNA methylation.

Figure 4.

Regional genomic plot for the coordinates surrounding the PIK3CD (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit δ) CpG sites (cg12033075 and cg03971555) on chromosome 1. The top plot contains the P values from the Cox proportional hazard analysis. Regulatory information related to promoter and TF (transcription factor) binding for this genomic region is shown, along with boundaries for genes in the region. The vertical gray dotted line passes through the genomic coordinates of the PIK3CD CpG sites to align with the regulatory and gene annotations. The DNase hypersensitivity (DHS) track highlights regions of DHS. The CTCF (CCCTC-binding factor) Binding Site track represents regions of CTCF binding. Additional details regarding the regulatory features are provided in the online supplement.