Pharmacogenetics (PGx) represents a component of precision medicine that enables individualized determina- tion of drug response. The benefits of PGx include reduced cost and risk of adverse drug reactions (SADRs), as well as improved drug efficacy. While there is a large number of PGx genes currently tested, Cytochrome P450 2D6 (CYP2D6) is of tremendous diagnostic value, as up to 25% of all drugs are activated or metabolized by CYP2D6. These drugs include cancer drugs, opioid agonists, and several antidepressants and antianxiety med- ications. The CYP2D6 enzyme is encoded by the CYP2D6 gene and genetic variation can cause a reduction or complete loss of enzyme function. CYP2D6 is primarily expressed in the liver and is a major contributor to hepatic drug metabolism and clearance. Problems with correctly diagnosing CYP2D6 genetic variation can directly affect the risk for the development of SADRs. There are over 140 described pharmacogenetic relevant variants (referred to as *star allele haplotypes) in CYP2D6, including frequent copy number variation. In addition, gene-fusions and hybrids with neighboring highly homologous (up to 94% identical) pseudogenes (CYP2D7 and CYP2D8) complicate variant calling. These fea- tures complicate genetic analysis with current testing platforms and many of the rare or more complex haplotypes are not accurately analyzed. Work from many groups, including ours, have demonstrated that currently used commercial genotyping platforms are prone to mischaracterize CYP2D6. This leads to incorrect assignment, which results in incorrect dosing recommendations. The goal of this application is to develop and evaluate an innovative approach to the sequence analysis of the CYP2D6 region in order to fully characterize the structure of CYP2D6. Our proposed approach is comprehensive yet scalable and can be performed with a turnaround time required for clinical testing. We propose to interrogate the full-length sequence, by using CRISPR-Cas9 technology to isolate the genomic locus and long-read se- quencing technologies. This approach, which we base on the preliminary data we present, has the potential to capture all potential haplotypes, including structural rearrangements and CNVs, with one assay. This application will serve as proof-of-concept that CRIPSR based sequence targeting, innovative fragment en- richment and single-molecule sequencing is a feasible approach. This will serve as the foundation for a Phase 2 grant application with the goals to expand the dataset, refine and optimize the protocol as a robust and accurate clinical platform and implement the test in a clinical testing environment.
This application focuses on a novel approach for accurate characterization of the CYP2D6 gene sequence. CYP2D6 characterization is of tremendous importance to many patients, as its structure affects the way patients respond to many drugs.
