The team led by the Pomeranian Medical University developed an epigenetic test based on DNA methylation analysis that distinguishes smokers from non-smokers — a breakthrough with potential applications in both medicine and forensic science.

The study results were published in Clinical Epigenetics and Toxicology and Applied Pharmacology.

According to Dr. Ewelina Pośpiech, the first author of the study, DNA methylation analysis can help profile an individual’s lifestyle from biological material collected during criminal investigations, complementing existing methods for creating a perpetrator profile.

“Assessing smoking status based on DNA methylation analysis can allow to profile the lifestyle of a person whose biological material is being analysed in a criminal case, complementing the profile of the perpetrator,” Pośpiech said.

The method also has medical applications. Patients often underreport their smoking habits, and the body’s response to cigarette smoke can vary.

“DNA methylation analysis provides objective data that reliably reflects the impact of smoking on DNA and the risk of developing smoking-related diseases,” Pośpiech said.

Her team analysed DNA methylation profiles in nearly 800 Polish men and women, identifying over 450 genomic sites affected by smoking. Four sites with the highest diagnostic value were selected for the predictive model, with the AHRR gene showing the strongest signal.

Unlike earlier studies, which focused primarily on blood samples, the Polish team also examined oral swabs.

“Meanwhile, forensics collects various types of biological material, and the DNA methylation pattern is specific to each tissue. It appears differently in blood than in saliva, semen, or bone,” Pośpiech said.“In medicine, it is also easier to use a patient’s saliva sample than to draw blood, for example, when we want to regularly monitor their compliance or how they are responding to therapy. Furthermore, saliva is directly exposed to cigarette smoke, so it produces a very strong epigenetic response. That is why we decided to expand the analyses to include oral swabs as well.”

Pośpiech added that the models’ compactness — analysing only a few genomic sites — is a major advantage in forensic laboratories, where DNA samples are often limited or degraded.

The study also showed that DNA methylation patterns vary with smoking intensity. Occasional and heavy smokers display distinct profiles, while former smokers’ profiles fall between those of current and never smokers.

“This is because DNA methylation is reversible. This means we can observe how a patient’s health returns to normal after quitting smoking. However, different markers take different amounts of time. In the case of the AHRR gene, this process is quite slow and can take more than five years,” Pośpiech said.

In the study, former smokers had quit on average 15 years earlier. Methylation of three of the four analysed markers had returned to normal, but the AHRR gene remained altered.

The researchers also discovered that AHRR gene methylation depends not only on cigarette smoke exposure but also on sleep duration and physical activity.

“This means that DNA methylation markers related to cigarette smoking can be influenced not only by quitting cigarettes but also by implementing other healthy habits. If someone quits smoking while also increasing their activity level and taking care of their sleep, the recovery process may be shorter,” Pośpiech said.

The predictive models developed by the Polish team have two key applications. In forensic practice, they can help analyse biological traces, build criminal profiles, and narrow suspect pools when there are no leads.

“If we do not have suspects in a criminal case, any additional information obtained from DNA that narrows the search scope is extremely valuable. Until now, we focused on predicting physical characteristics, but now, thanks to DNA methylation, we can also talk about behaviours, for example, whether a person smokes cigarettes,” Pośpiech said.

In medicine, the tool provides a reliable way to verify smoking status and assess physiological impact.

“We can, of course, ask a patient if they smoke, but we do not always get an honest answer, which makes it difficult to tailor appropriate treatment. Furthermore, everyone reacts differently to the same dose of tobacco smoke. DNA methylation analysis allows to objectively assess whether and how strongly the body reacts to cigarettes, enabling us to more effectively monitor the patient, check their compliance, and assess the risk of developing diseases related to this addiction, such as cardiovascular disease or lung cancer,” Pośpiech said.
“This is of enormous importance in preventive medicine. Smoking is one of the most important causes of mortality in the population. If we monitor methylation markers and respond in a timely manner, we will be able to significantly reduce the risk of death,” she added.

The same approach strengthens epidemiological studies, in which participants do not always disclose their addictions. Predictive models offer objective, verifiable data that improve research accuracy.

Scientists from the Pomeranian Medical University in Szczecin, the Jagiellonian University, the Central Forensic Laboratory of the Police, the University of Lodz, and the Academy of Physical Education in Katowice collaborated on the project.

The study was funded by the Polish National Centre for Research and Development and ABB.

Katarzyna Czechowicz (PAP)

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