{"doi":"10.3389/fphar.2024.1429286","title":"CYP2D6 copy number determination using digital PCR","abstract":"Background CYP2D6 testing is increasingly used to guide drug therapy and thus, reliable methods are needed to test this complex and polymorphic gene locus. A particular challenge arises from the detection and interpretation of structural variants (SVs) including gene deletions, duplications, and hybrids with the CYP2D7 pseudogene. This study validated the Absolute Q ™ platform for digital PCR-based CYP2D6 copy number variation (CNV) determination by comparing results to those obtained with a previously established method using the QX200 platform. In addition, protocols for streamlining CYP2D6 CNV testing were established and validated including the “One-pot” single-step restriction enzyme digestion and a multiplex assay simultaneously targeting the CYP2D6 5′UTR, intron 6, and exon 9 regions. Methods Genomic DNA (gDNA) samples from Coriell (n = 13) and from blood, saliva, and liver tissue (n = 17) representing 0–6 copies were tested on the Absolute Q and QX200 platforms. Custom TaqMan™ copy number (CN) assays targeting CYP2D6 the 5′UTR, intron 6, and exon 9 regions and a reference gene assay ( TERT or RNaseP) were combined for multiplexing by optical channel. In addition, two digestion methods (One-pot digestion and traditional) were assessed. Inconclusive CN values on the Absolute Q were resolved using an alternate reference gene and/or diluting gDNA. Results Overall, results between the two platforms and digestions methods were consistent. The “One-pot” digestion method and optically multiplexing up to three CYP2D6 regions yielded consistent result across DNA sample types and diverse SVs, reliably detecting up to 6 gene copies. Rare variation in reference genes were found to interfere with results and interpretation, which were resolved by using a different reference. Conclusion The Absolute Q produced accurate and reliable CYP2D6 copy number results allowing for a streamlined and economical protocol using One-pot digestion and multiplexing three target regions. Protocols are currently being expanded to other pharmacogenes presenting with SVs/CNVs.","journal":"Frontiers in Pharmacology","year":2024,"id":445290,"datarank":0.31191623125197543,"base_score":2.0794415416798357,"endowment":2.0794415416798357,"self_citation_contribution":0.31191623125197543,"citation_network_contribution":0.0,"self_endowment_contribution":0.31191623125197543,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":7,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9587,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1261244,"name":"L S Lin","orcid":"0009-0005-4962-8370","position":1,"is_corresponding":false},{"id":442252,"name":"Erin C. Boone","orcid":"0000-0003-3044-5521","position":2,"is_corresponding":false},{"id":1261735,"name":"Junko Stevens","orcid":null,"position":3,"is_corresponding":false},{"id":232876,"name":"Andrea Gaedigk","orcid":"0000-0001-6968-1893","position":4,"is_corresponding":false},{"id":442253,"name":"Wendy Y. Wang","orcid":"0000-0002-0203-0196","position":0,"is_corresponding":true}],"reference_count":29,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:01:42.089235Z","pmid":"39206265","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}