In summary, epigenetic biomarkers for smoking appear to meet the key criteria for potential successful clinical translation, particularly at the two sites with the most replicated associations, AHRR and F2RL3. Among the studies reviewed above, methodological issues are adequately addressed, including controlling for batch effects, cell mixture, confirming exposure with other biomarkers such as cotinine, controlling for other exposures such as cannabis, and most importantly performing careful phenotyping. The studies above include appropriate sensitivity and specificity analyses, demonstrate dose-response relationships between exposure and methylation, and capture both persistent signatures of past smoking and reversible signatures that indicate cessation time. In addition, these loci demonstrate the ability to predict important clinical outcomes such as mortality. In terms of generalizability, the studies include populations of different ages, ethnicities, sexes, use patterns, and periods of developmental exposure. Of note, findings at AHRR and F2RL3 did not replicate consistently across non-blood tissues, indicating that other loci may be more appropriate if other tissue sources are to be used clinically. However, on the whole, this review found sufficient evidence to recommend development of epigenetic biomarkers for smoking as clinical tools with the potential for tremendous public health impact.
