1 The world leader in serving science Toinette Hartshorne, Ph.D. Sr. Staff Applications Scientist, Genetic Analysis Genetic, Medical & Applied Sciences Thermo Fisher Scientific CYP2D6 Allele Specific Copy Number Analysis Using TaqMan® SNP Genotyping Assays And Digital PCR
17
Embed
CYP2D6 Allele Specific Copy Number Analysis Using TaqMan® SNP Genotyping Assays And Digital PCR
This presentation provides an overview of performing allele-specific copy number variation (CNV) analysis using digital PCR. We demonstrate a pharmacogenomics analysis workflow using the QuantStudio® 12k Flex system to perform CYP2D6 SNP genotyping and copy number analysis, followed by allele-specific copy number analysis using TaqMan® SNP Genotyping Assays on the QuantStudio® 3D digital PCR system. Digital PCR enables precise determination of the specific allele composition of samples with duplications in the CYP2D6 gene, which can impact the extent to which certain drugs are metabolized.
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Likely phenotype Activity score Genotypes Examples of
diplotypes Ultrarapid metabolizer >2.0 more than two copies of functional alleles *1/*1x2,
*1/*2x2
Extensive metabolizer 1.0-2.0
two full or reduced function alleles or
one full function allele plus either one nonfunctional or one reduced function allele
*1/*1, *1/*2, *10/*10,
*1/*4, *10/*5Intermediate metabolizer 0.5 one reduced and one nonfunctional allele *4/*10,
*5/*17
Poor metabolizer 0 no functional alleles *4/*4, *4/*5, *5/*5, *4/*6
Predicted metabolizer phenotype is used to determine starting drug dosages.
E.g., this chart is relevant to the metabolism of codeine to morphine by CYP2D6:
• Ultrarapid metabolizers avoid codeine use due to potentially toxic morphine levels. • Poor metabolizers avoid codeine use due to lack of efficacy. • Extensive & Intermediate metabolizers use age- & weight-specific dosing
• Intermediate metabolizers may not respond as well as extensive metabolizers
CYP2D6 Diplotype Determines Drug Metabolism
table data from: www.pharmgkb.org
7
Phenotypic Outcomes Can Vary Depending On Duplicated Allele In Heterozygous Individuals
*1 / *10 x 2Extensive
metabolizer
*1 x 2 / *10Ultrarapid
metabolizer
*10 reduced function allele *1 full function allele
*4 / *17 x 2Extensive
metabolizer
*4 x 2 / *17Intermediate metabolizer
*17 reduced function allele *4 nonfunctional allele
Metabolizer status will depend on which allele has
been duplicated
~1-2% duplicated alleles
heterozygous for different functional
classesE
xam
ple
1E
xam
ple
2
8
Identify the duplicated allele to enable more accurate drug metabolizer status prediction
OpenArray® Plate Genotyper™ Software
SNP Genotyping Analysis
96- or 384-well Plate CopyCaller® Software
Copy Number Analysis
AlleleTyper™ Software
Star Allele Results
*NEW* Allele-specific Copy Number Analysis
QuantStudio® 3D Digital PCR System
TaqMan® SNP Assays
TaqMan® Pharmacogenomics Experiment Workflow
+
~1-2% of samples
9
How Digital PCR Works
Digital PCR is an analytical technique for quantification of nucleic acid samples based on PCR amplification of single template molecules
Negative reactions
Preparation DistributionCount
Negatives
gDNA or cDNATaqMan® AssayTaqMan® Master Mix
PCR Reaction
Output:# of molecules/µL
10
CYP2D6 Allele-Specific Copy Number dPCR Workflow
AmplifyLoad ReadMix
QuantStudio 3DAnalysisSuite™ Software
SpeI
1. Identify samples with CYP2D6 duplications that are heterozygous for functionally different alleles
Purpose: alleles can be distributed to separate wells
4. Analyze data o Review & confirm cluster plots
o Calculate VIC® and FAM™ dye ratios using data from AnalysisSuite™
2. Digest gDNAs to separate tandem duplicated alleles
3. Run digested material with appropriate TaqMan® SNP Assays
Allele-specific copy number analysis using dPCR & TaqMan® SNP Genotyping Assays is a simple & effective method for identifying specific duplicated alleles in heterozygous samples
This method facilitates accurate CYP2D6 allele genotyping & better prediction of drug metabolizer phenotype.
Other applications for allele-specific copy number analysis include:
Genomic targets where CNV of particular alleles is functionally important