Simple and Robust Detection of <i>CYP2D6</i> Gene Deletions and Duplications Using <i>CYP2D8P</i> as Reference

• Main Authors: Jens Borggaard Larsen (Author), Steffen Jørgensen (Author)
• Format: Book
• Published: MDPI AG, 2022-01-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.
• Notes: 10.3390/ph15020166; 1424-8247
• Keywords: <i>CYP2D6</i>; <i>CYP2D7P</i>; <i>CYP2D8P</i>; copy number variation; CNV; genotyping; Medicine; R; Pharmacy and materia medica; RS1-441
• Source: Pharmaceuticals, Vol 15, Iss 2, p 166 (2022)

Genotyping of the <i>CYP2D6</i> gene is the most commonly applied pharmacogenetic test globally. Significant economic interests have led to the development of a plurality of assays, available for almost any genotyping platform or DNA detection chemistry. Of all the genetic variants, copy number variations are particular difficult to detect by polymerase chain reaction. Here, we present two simple novel approaches for the identification of samples carrying either deletions or duplications of the <i>CYP2D6</i> gene; by relative quantification using a singleplex 5'nuclease real-time PCR assay, and by high-resolution melting of PCR products. These methods make use of universal primers, targeting both the <i>CYP2D6</i> and the reference gene <i>CYP2D8P</i>, which is necessary for the analysis. The assays were validated against a reference method using a large set of samples. The singleplex nature of the 5'nuclease real-time PCR ensures that the primers anneal with equal affinity to both the sequence of the <i>CYP2D6</i> and the reference gene. This facilitates robust identification of gene deletions and duplications based on the cycle threshold value. In contrast, the high-resolution melting assay is an end-point PCR, where the identification relies on variations between the amount of product generated from each of the two genes.