Association between DNA methylation and smoking in patients with chronic limb-threatening ischemia

Introduction and objective: to study the association between smoking and the methylation level of 2 genomic regions in patients with peripheral artery disease (PAD).Method: cross-sectional study of 297 patients (mean age, 69.6 years; males, 78.5%) diagnosed with chronic lower extremity ischemia at various clinical stages from March 2016 through December 2019 at the Vascular Surgery Unit of Hospital del Mar, Barcelona, Catalonia, Spain. Methylation analysis of Cg02156642 and Cg03636183, previously associated with smoking in former studies was performed. Multiple linear regression was conducted to identify variables associated with methylation levels. The area under the ROC curve was estimated to discriminate meth-ylation levels between smokers and non-smokers.Results: among the sample, 46 patients (15.5%) were non-smokers, 132 (44.4%) were former smokers, and 119 (40.1%) were current smokers. No association was seen between tobacco exposure and methylation levels of Cg02156642. However, an association was found with Cg03636183: smokers had lower methylation levels, and a higher smoking load was associated with lower methylation (Spearman's Rho,-0.324; p < .001). A methylation level of 80% in this region showed a 90.0% sensitivity and an 83.5% specificity to discriminate between smokers and never smokers. To discriminate between smokers and former smokers, a methylation level of 75% had an 69% sensitivity and an 56.9% specificity. After adjusting for all variables associated with methylation, the association between Cg03636183 and smoking remained significant among never smokers and smokers.Conclusions: methylation of the Cg03636183 region is associated with smoking in patients with PAD and is directly associated with the smoking load. This biomarker could be used in the routine clinical practice to assess tobacco use in our patients.