Increased resolution screening of the pharmacogenetic gene CYP2D6 with microarray technology
Davis, Carey P.
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Autopsy is a primary methodology used for assessing cause and/or manner of death in medicolegal investigations. Some autopsies, however, do not resolve the cause of death unambiguously or there is no evident pathology to determine the cause of death. In addition, in some cases toxicology screens are negative or difficult to interpret because it is challenging to determine if a high concentration of a drug in the body derived from one large dose or has built up over time. Determining the genetic constitution of victims at specific target genes may clarify the cause of some of these unexplained deaths or at least indicate susceptibility to triggering effects. Cytochrome P450 (CYP450) is a super family of enzymes that detoxify foreign chemicals and are involved in the metabolism of drugs. One gene in this family that encodes CYP450 enzymes, CYP2D6, accounts for the metabolism of 25% of all drugs currently on the market. By examining the variability in the CYP2D6 gene, a SNP panel was developed and used to aid in personalized medicine with a long-term outcome of reducing risk in patients who partake in drug therapy. However, paralogs of the CYP2D6 gene can interfere with obtaining accurate typing results. The hypothesis of this dissertation is that it is possible to develop a targeted panel for clinically relevant variants in the CYP2D6 gene using array-based technology that can provide accurate and reliable genotyping results. The goal was first to demonstrate that high throughput sequencing, also known as next generation or massively parallel sequencing, could reliably sequence a complex target using a model system, i.e. the hypervariable regions of the human mitochondrial genome. Then, using this advanced sequencing capability define the baseline genetic variation of the CYP2D6 gene in a selected population and identify those genetic markers associated with metabolism capacity that would be verified against a database of actionable variants that cause reaction to drug exposure. The entire CYP2D6 gene was sequenced to identify SNPs at a population level. These SNPs were compared against a known database of clinically relevant samples with known metabolic responses of the same ethnic background to verify actionable variants. Once these variants were identified, a PCR assay workflow leveraging microarray technology was developed to quickly and efficiently screen individuals of interest by overcoming paralog interference. This assay can be used prior to administering drugs or post mortem to gain information about potential adverse drug reactions.