The fourth approach is to identify AUD-specific DNA methylation changes using a well-characterized and well-powered sample of AUD cases, and matched healthy controls. DNA methylation status is affected by the frequency or severity of alcohol use.37 Instead of relying on self-report measures of alcohol use, ascertaining alcohol use status by treatment setting (ie, inpatient detoxification) or measuring blood alcohol levels for characterizing AUD status may be advantageous for examination of AUD-associated DNA methylation alterations. However, this also carries the substantial limitation of dosage ascertainment, and only a limited number of inspections are conducted over a lifetime or even across generations, while epigenetic marks are more likely to develop over the long term. Additionally, AUD subjects often have comorbid smoking, drug use, or psychiatric disorders (such as schizophrenia, bipolar disorders, and depression).110 It is necessary to clarify whether DNA methylation changes observed in AUD subjects are attributable to alcohol consumption or due to comorbid nicotine or drug use, or other psychiatric disorders. When analyzing AUD-associated DNA methylation changes, we can exclude those subjects who use illicit drugs of abuse or are
