Estimating the Associations of Regular Cannabis Use with Epigenetic Ageing and Epigenetic-G in Adults Versus Adolescents

This study examines whether regular cannabis use is associated with differences in DNA methylation (DNAm)-derived indicators of biological aging and cognitive function, and whether these associations differ between adolescents and adults. The project uses data from the CannTeen study, a longitudinal observational cohort designed to investigate developmental differences in the effects of regular cannabis use. Cannabis is one of the most widely used psychoactive substances globally, with initiation frequently occurring during adolescence. Previous research suggests that adolescence may represent a period of increased vulnerability to cannabis-related harms, including greater risk of cannabis use disorder, cognitive difficulties, and adverse mental health outcomes. However, the biological mechanisms that may underlie these developmental differences remain poorly understood. DNA methylation provides a promising approach for investigating how environmental exposures such as cannabis use become biologically embedded and may influence processes related to aging, cognition, and health. This study focuses on three DNAm-derived biomarkers. First, epigenetic age acceleration (AgeAccel) will be assessed using established epigenetic clocks that estimate biological age relative to chronological age. Second, DunedinPACE will be used to measure the pace of biological aging, reflecting the rate at which physiological decline is occurring. Third, Epigenetic-g, a DNA methylation-derived proxy of general cognitive ability, will be used as a molecular indicator of cognitive functioning. Together, these biomarkers provide complementary measures of biological aging, aging rate, and cognitive performance. Participants include adolescents aged 16–17 years and adults aged 26–29 years recruited as part of the CannTeen study. Individuals who regularly use cannabis are compared with non-using controls, creating a 2 × 2 design crossing age group (adolescent versus adult) and cannabis use status (regular use versus control). Saliva DNA methylation was measured using the Illumina HumanMethylationEPIC BeadChip array. Data have undergone extensive quality control and preprocessing prior to biomarker calculation. The primary objective is to test whether regular cannabis use is associated with epigenetic indicators of accelerated aging and reduced cognitive performance, and whether these associations are stronger during adolescence than adulthood. Three preregistered hypotheses will be evaluated. First, regular cannabis use is expected to be associated with greater epigenetic age acceleration, with larger effects observed among adolescents. Second, regular cannabis use is expected to be associated with a faster pace of biological aging as measured by DunedinPACE, with stronger associations in adolescents. Third, regular cannabis use is expected to be associated with lower Epigenetic-g scores, indicating poorer methylation-derived cognitive functioning, with larger differences in adolescents than adults. Primary analyses will use linear regression models that include age group, cannabis use status, and their interaction, while adjusting for sex, saliva cell composition estimates, technical batch variables, and tobacco exposure. The AgeGroup × Cannabis interaction is the primary test of developmental sensitivity, allowing assessment of whether cannabis-related epigenetic differences are more pronounced during adolescence than adulthood. Expected outcomes are that regular cannabis use will be associated with older epigenetic age profiles across aging and weaker epigenetic-associated cognitive domains, and that these associations will be strongest among adolescents when compared to adults. Findings will contribute to understanding whether adolescence represents a period of heightened biological sensitivity to cannabis exposure and may provide insight into potential molecular pathways linking cannabis use with long-term health and cognitive outcomes. More broadly, the study aims to advance knowledge of how cannabis use relates to biological aging and cognitive function across different developmental stages.