title: Flexible and Scalable Full-Length CYP2D6 Long Amplicon PacBio Sequencing.
abstract: Cytochrome P450 2D6 (CYP2D6) is among the most important genes involved in drug metabolism. Specific variants are associated with changes in the enzyme's amount and activity. Multiple technologies exist to determine these variants, like the AmpliChip CYP450 test, Taqman qPCR, or Second-Generation Sequencing, however, sequence homology between cytochrome P450 genes and pseudogene CYP2D7 impairs reliable CYP2D6 genotyping, and variant phasing cannot accurately be determined using these assays. To circumvent this, we sequenced CYP2D6 using the Pacific Biosciences RSII and obtained high-quality, full-length, phased CYP2D6 sequences, enabling accurate variant calling and haplotyping of the entire gene-locus including exonic, intronic, and upstream and downstream regions. Unphased diplotypes (Roche AmpliChip CYP450 test) were confirmed for 24 of the 25 samples, including gene duplications. Cases with gene deletions required additional specific assays to resolve. In total, 61 unique variants were detected, including variants that had not previously been associated with specific haplotypes. To further aid genomic analysis using standard reference sequences, we have established an LOVD-powered CYP2D6 gene-variant database, and added all reference haplotypes and data reported here. We conclude that our CYP2D6 genotyping approach produces reliable CYP2D6 diplotypes and reveals information about additional variants, including phasing and copy-number variation.
authors.affiliation: Leiden Genome Technology Center, Department of Human Genetics, Leiden University Medical Center, Leiden, 2333ZC, The Netherlands.
authors.forename: Henk P J
authors.initials: HP
authors.lastname: Buermans
authors.affiliation: Leiden Genome Technology Center, Department of Human Genetics, Leiden University Medical Center, Leiden, 2333ZC, The Netherlands.
authors.forename: Rolf H A M
authors.initials: RH
authors.lastname: Vossen
authors.affiliation: Leiden Genome Technology Center, Department of Human Genetics, Leiden University Medical Center, Leiden, 2333ZC, The Netherlands.
authors.forename: Seyed Yahya
authors.initials: SY
authors.lastname: Anvar
authors.affiliation: Leiden Genome Technology Center, Department of Human Genetics, Leiden University Medical Center, Leiden, 2333ZC, The Netherlands.
authors.forename: William G
authors.initials: WG
authors.lastname: Allard
authors.affiliation: Department of Clinical Pharmacy & Toxicology, Leiden University Medical Center, Leiden, 2333ZA, The Netherlands.
authors.forename: Henk-Jan
authors.initials: HJ
authors.lastname: Guchelaar
authors.affiliation: Leiden Genome Technology Center, Department of Human Genetics, Leiden University Medical Center, Leiden, 2333ZC, The Netherlands.
authors.forename: Stefan J
authors.initials: SJ
authors.lastname: White
authors.affiliation: Leiden Genome Technology Center, Department of Human Genetics, Leiden University Medical Center, Leiden, 2333ZC, The Netherlands.|Department of Clinical Genetics, Leiden University Medical Center, Leiden, 2333ZC, The Netherlands.
authors.forename: Johan T
authors.initials: JT
authors.lastname: den Dunnen
authors.affiliation: Department of Clinical Pharmacy & Toxicology, Leiden University Medical Center, Leiden, 2333ZA, The Netherlands.
authors.forename: Jesse J
authors.initials: JJ
authors.lastname: Swen
authors.affiliation: Department of Clinical Pharmacy & Toxicology, Leiden University Medical Center, Leiden, 2333ZA, The Netherlands.
authors.forename: Tahar
authors.initials: T
authors.lastname: van der Straaten
mesh_terms: D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D056915:DNA Copy Number Variations; D017353:Gene Deletion; D020440:Gene Duplication; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D017422:Sequence Analysis, DNA; D014178:Translocation, Genetic
chemical_list: D019389:Cytochrome P-450 CYP2D6
keywords: CYP2D6; PacBio long-read sequencing; copy-number variation; pharmacogenomics; variant phasing