Cannabis use is an important global public health issue, and a growing topic of controversy and debate1,2. It is the most widely used illicit psychoactive substance in the world3, and the potential medicinal and therapeutic benefits of cannabis and its main active ingredients tetrahydrocannabinol (THC) and cannabidiol (CBD) are gaining interest4,5,6. There is strong evidence to suggest that the heavy and prolonged use of cannabis may be associated with increased risk of adverse outcomes in a number of areas, including mental health (psychosis7,8,9, schizophrenia10,11, depression12,13) and illicit drug abuse14.

Drug metabolism, drug response and drug addiction have known genetic components15, and multiple genome-wide association studies (GWAS) have identified genes and allelic variants that are likely contributors to substance use disorders16,17. There are aspects of cannabis use disorder that are heritable18,19,20,21, and several candidate loci for complex phenotypes such as lifetime cannabis use have recently been identified3,22 that explain a proportion of the variance in cannabis use heritability. Complex phenotypes like these are influenced by multiple loci, each of which usually has a small individual effect size23, and such loci are frequently located in non-coding regions of the genome24,25, making their biological role difficult to elucidate.

Epigenetic mechanisms are involved in the interaction between the genome and environment; they respond to changes in environmental stimuli (such as diet, exercise, drugs), and act to alter chromatin structure and thus regulate gene expression26. Epigenetic modifications, such as DNA methylation, contribute to complex traits and diseases27,28. Methylation of cytosine residues within CpG dinucleotides is an important mechanism of variation and regulation in the genome29,30,31,32. Cytosine methylation, particularly in the promoter region of genes, is often associated with a decrease in transcription33, and DNA methylation in the first intron and gene expression is correlated and conserved across tissues and vertebrate species34. Furthermore, modulation of methylation at CpG sites within the human genome can result in an epigenetic pattern that is specific to individual environmental exposures, and these may contribute to disease26,35,36,37. For example, environmental factors such as drugs, alcohol, stress, nutrition, bacterial infection, and exercise36,38,39,40,41 have been associated with methylation changes. A number of these methylation changes have been shown to endure and induce lasting biological changes36, whereas others are dynamic and transient. For example, alcohol consumption affects genome-wide methylation patterns in a severity-dependent manner42 and some of these changes revert upon abstinence from alcohol consumption43. A similar observation is reported for former tobacco smokers, with DNA methylation changes after cessation eventually reaching levels close to those who had never smoked tobacco44. Thus, DNA methylation can be indicative of a particular environmental exposure, shed light on the dynamic interaction between the environment and the genome, and provide new insights in to the biological response.

Recreational drug use (an environmental stimulus) has been associated with adverse mental health outcomes, particularly in youth45,46,47,48,49, and epigenetics may play a role in mediating the biology involved. Therefore, we sought to determine whether regular cannabis users displayed differential cytosine methylation compared with non-cannabis users. Cannabis users in this study are participants from the Christchurch Health and Development Study (CHDS), a longitudinal study of a birth cohort of 1265 children born in 1977 in Christchurch, New Zealand. Users often consume cannabis in combination with tobacco. Unusually, the CHDS cohort contains a subset of cannabis users who have never consumed tobacco, thus enabling an investigation of the specific effects of cannabis consumption, in isolation, on DNA methylation in the human genome.