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Single-Cell Sequencing Study Finds Increased Single-Nucleotide Mutation Rate in Lungs of Smokers

NEW YORK — Mutations accumulate in people's lungs as they age, but more mutations arise in the lungs of smokers as they get older, and depending on how much they smoke, a new single-cell analysis has found.

Smoking has long been thought to dial up lung cancer risk by increasing the number of mutations in lung cells. By sequencing lung cells from people who never smoked as well as smokers, a team led by researchers at Albert Einstein College of Medicine has now confirmed that mutation frequencies do tend to be higher in lung cells of smokers. In a study published in Nature Genetics on Monday, they showed that this increase in mutation rate plateaued after 23 pack-years of exposure — with a pack-year defined as 20 cigarettes smoked each day for a year — suggesting that there could be mechanisms that suppress additional mutation accumulation and lung cancer development.

"The heaviest smokers did not have the highest mutation burden," co-senior author Simon Spivack, a professor of medicine and genetics at Einstein and a pulmonologist at the Montefiore Health System, said in a statement. "Our data suggest that these individuals may have survived for so long in spite of their heavy smoking because they managed to suppress further mutation accumulation. This leveling off of mutations could stem from these people having very proficient systems for repairing DNA damage or detoxifying cigarette smoke."

The researchers used a single-cell multiple displacement amplification approach to analyze normal proximal bronchial basal cells from 19 smokers, including both former and current smokers, and from 14 never-smokers, including two adolescents. They analyzed between three and eight nuclei per participant and looked at the number of single-nucleotide variants and indels in those samples.

Overall, for never-smokers, the median number of SNVs per nucleus ranged from about 464 for an 11 year-old to about 2,739 for an 86-year-old, translating to a rate of 28 mutations per cell per year. The median number of indels likewise increased with age, ranging from about 59 to 304, corresponding to a rate of about two indels per cell per year.

Smokers, though, had a higher SNV mutation rate of about 91 SNVs per cell per year, an excess of 63 SNVs per cell per year over never-smokers. They had similar indel mutation rates as non-smokers, though.

"This experimentally confirms that smoking increases lung cancer risk by increasing the frequency of mutations, as previously hypothesized," Spivack said. "This is likely one reason why so few non-smokers get lung cancer, while 10 percent to 20 percent of lifelong smokers do."

However, that increase in mutation rate eventually levels off, the researchers found. By examining mutation frequency as a function of cumulative smoking dose — calculated through pack-years — the researchers found that the mutation frequency among smokers increased linearly until 23 pack-years, after which it remained constant.

This, the researchers noted, could account for why heavy smokers have similar mutational burdens as lighter smokers. They further suggested that there could be increased resilience among some individuals against the accumulation of new mutations, noting that two study participants were carriers of an AKR1C2 polymorphism that helps with the detoxification of the polycyclic aromatic hydrocarbons that are found in cigarette smoke and contribute to mutation formation.

The results also open up a new avenue for research. Jan Vijg, a study co-senior author and professor at Einstein, noted in a statement that he and his colleagues now plan to develop assays to gauge DNA repair or detoxification ability.