Development of a Triplex FMCA Assay for Genotyping Three Genes, ADH1B, ADH1C, and ALDH2, Involved in Alcohol Metabolism

Three functional single nucleotide variants (SNVs)‒ ALDH2 rs671 (p.E504K), ADH1C rs698 (p.I350V), and ADH1B rs1229984 (p.R48H)‒are key genetic determinants of human alcohol metabolism. These variants significantly affect drinking behavior and are associated with liver disease and increased risks of several malignancies, including esophageal and gastric cancers. We developed a triplex fluorescent probe-based melting curve analysis (FMCA) assay for the simultaneous detection of these three SNVs. The assay was validated by comparing FMCA results with Sanger sequencing using genomic DNA from 94 Japanese individuals. The automated detection algorithm reliably identified genotypes of rs671 and rs698. Although the melting peaks of rs1229984 exhibited lower resolution and necessitated manual visual inspection for definitive genotype discrimination, all genotypes were nevertheless correctly identified. The assay demonstrated 100% accuracy. In conclusion, this triplex FMCA assay provides a rapid, cost-effective, and streamlined method for the simultaneous genotyping of ADH1B , ADH1C , and ALDH2 . Given its high accuracy and ease of implementation, this method serves as a practical alternative to conventional sequencing, positioning it as a valuable tool for both large-scale epidemiological research and routine clinical assessment of alcohol-related health risks.