CYP2D6 Allele Specific Copy Number Analysis Using TaqMan® SNP Genotyping Assays And Digital PCR
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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.
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CYP2D6 Allele Specific Copy Number Analysis Using TaqMan® SNP Genotyping Assays And Digital PCR
- 1. CYP2D6 Allele Specific Copy Number Analysis Using TaqMan® SNP Genotyping Assays And Digital PCR The world leader in serving scien1ce Toinette Hartshorne, Ph.D. Sr. Staff Applications Scientist, Genetic Analysis Genetic, Medical & Applied Sciences Thermo Fisher Scientific
- 2. 2 What Is Pharmacogenomics? • Pharmacogenomics (PGx) is the study of genetic variation that determines how individuals respond to specific drugs • It allows physicians to more accurately determine the right drug and dosage for the patient (avoid adverse drug reactions (ADRs)) • Becoming increasingly important for pain treatment and psychotropic and cardiac drug dose management Figure from www.pharmainfo.net/reviews/role-pharmacogenomics-drug-development.
- 3. 3 PGx Drug Metabolism Enzymes (DMEs) DMEs catalyze reactions that affect the absorption, distribution, metabolism, excretion of drugs Cytochrome P450 system • Phase I metabolic system of the liver • Metabolism of >85% of medications • Genetic variability affects pharmacokinetics
- 4. Genotyping Highly Polymorphic CYP2D6 Is Challenging • > 100 characterized CYP2D6 ‘star’ alleles that can contain multiple polymorphisms • Genotype analysis requires both SNP genoptyping and CNV analysis 4 • > 80 known CYP2D6 polymorphisms within coding and regulatory regions • includes SNPs, InDels, CNVs, and gene conversion events Figure adapted from Nature Reviews Drug Discovery (2004)
- 5. 5 CYP2D6 Star Allele Haplotypes Can Be Duplicated • Star (*) alleles are gene-level haplotypes that are associated with DME phenotypes • For phenotype interpretation purposes, genotyping results are translated to star allele nomenclature • The following CYP2D6 star alleles can be duplicated in individuals: haplotype Major SNPs Enzyme Function Activity score *1 reference Full 1.0 *2 2850C>T; 4180G>C Full 1.0 *2A -1584C>G; 2850C>T; 4180G>C Full 1.0 *4 100C>T; 1846G>A; 4180G>C None 0 *9 2615_2617delAAG Reduced 0.5 *10 100C>T; 4180G>C Reduced 0.5 *17 1023C>T; 2850C>T; 4180G>C Reduced 0.5 *35 -1584C>G; 31G>A; 2850C>T; 4180G>C Full >=1.0 table data from: www.cypalleles.ki.se
- 6. 6 CYP2D6 Diplotype Determines Drug Metabolism 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/*5 Intermediate 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 table data from: www.pharmgkb.org
- 7. 7 Phenotypic Outcomes Can Vary Depending On Duplicated Allele In Heterozygous Individuals *10 reduced function allele *1 full function allele *1 / *10 x 2 Extensive metabolizer *1 x 2 / *10 Ultrarapid metabolizer *17 reduced function allele *4 nonfunctional allele *4 / *17 x 2 Extensive metabolizer *4 x 2 / *17 Intermediate metabolizer ~1-2% duplicated alleles heterozygous for different functional classes Metabolizer status will depend on which allele has been duplicated Example 2 Example 1
- 8. 8 TaqMan® Pharmacogenomics Experiment Workflow SNP Genotyping Analysis OpenArray® Plate Genotyper™ Software Copy Number Analysis 96- or 384-well Plate CopyCaller® Software Star Allele Results AlleleTyper™ Software *NEW* Allele-specific Copy Number Analysis Identify the duplicated allele to enable more accurate drug metabolizer status prediction QuantStudio® 3D Digital PCR System TaqMan® SNP Assays + ~1-2% of samples
- 9. 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 Distribution Count Negatives gDNA or cDNA TaqMan® Assay TaqMan® Master Mix PCR Reaction Output: # of molecules/μL
- 10. CYP2D6 Allele-Specific Copy Number dPCR Workflow 10 Mix Load Amplify Read QuantStudio® 3D AnalysisSuite™ Software SpeI 1. Identify samples with CYP2D6 duplications that are heterozygous for functionally different alleles Purpose: alleles can be distributed to separate wells 2. Digest gDNAs to separate tandem duplicated alleles 3. Run digested material with appropriate TaqMan® SNP Assays 4. Analyze data o Review & confirm cluster plots o Calculate VIC® and FAM™ dye ratios using data from AnalysisSuite™
- 11. TaqMan® Drug Metabolism Genotyping Assays tested 11 VIC® SNP/DME assay rs number common name C__32407252_30 rs1080985 CYP2D6*2A g. -1584C>G C__27102414_10 rs1135840 CYP2D6*2 g.4180G>C C__27102425_10 rs16947 CYP2D6*2 g.2850C>T C__27102431_D0 rs3892097 CYP2D6*4 g.1846G>A C__32407229_60 rs72549350 CYP2D6*9 g.2613 2615delAGA C__11484460_40 rs1065852 CYP2D6*10 g.100C>T C___2222771_A0 rs28371706 CYP2D6*17 g.1023C>T C__27102444_80 rs769258 CYP2D6*35 g.31G>A
- 12. 12 Test Coriell gDNA Samples of Known Genotypes (2 or 3 copies of CYP2D6) Samples Copy Number Genotype Target SNPs Expected Ratios (FAM:VIC) NA10859 2 *1/*2A 4180G>C 1:1 *2A -1584C>G 1:1 NA17116 2 *4/*17 *17 1023C>T 1:1 *10 100C>T 1:1 2850C>T 1:1 *4 1846G>A 1:1 NA17227 2 *1/*9 *9 delAGA 1:1 NA17105 3 *4x2/*70 *10 100C>T 1:2 2850C>T 2:1 *4 1846G>A 2:1 NA17117 3 *4x2/*10, *4/*10x2 *10 100C>T 0:1 *4 1846G>A 1:2 or 2:1 NA17155 3 *2x2/*17, *2/*17x2 *17 1023C>T 1:2 or 2:1 4180G>C 1:0 2850C>T 0:1 NA17209 2 + *36 *1/*4-*36 *10 100C>T 1:2 *4 1846G>A 2:1 4180G>C 1:1 NA17232 3 *2Ax2/*35, *2A/*35x2 *35 31G>A 1:2 or 2:1
- 13. 13 T Examples of dPCR Clusters Produced with 2 SNP Assays Run on a 2 Copy and a 3 Copy Sample C T C T A A G G empty empty empty empty T/C T/C A/G A/G
- 14. *17 1023C>T 1 of 3 1 of 2 0 of 3 2 of 3 1 of 2 3 of 3 1 of 2 1 of 2 NA17116 NA17105 NA17155 NA10859 NA17209 NA17155 NA17116 NA17155 14 Accurate Calling of Copy Number Ratios 120 100 80 60 40 20 0 FAM % FAM % Average 48.48 66.09 0.49 47.42 53.80 99.46 51.08 33.39 FAM % Expected 50.00 66.66 0.00 50.00 50.00 100.00 50.00 33.33 1 of 2 1 of 3 1 of 2 1 of 3 0 of 3 *4 1846G>A 2 of 3 2 of 3 2 of 3 NA17116 NA17105 NA17209 NA17117 NA17116 NA17209 NA17105 NA17117 70 60 50 40 30 20 10 0 FAM % FAM % Average 49.21 64.69 64.17 65.29 50.56 33.29 33.28 0.36 FAM % Expected 50.00 66.66 66.66 66.66 50.00 33.33 33.33 0.00
- 15. 15 Summary 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 Allele-specific gene expression analysis
- 16. 16 Acknowlegements R&D Sunali Patel Kathleen Hayashibara Trish Hegerich David Keys Kamini Varma Product Management Iain Russell Elliot Shelton Paco Cifuentes Thermo Fisher Scientific
- 17. 17 Legal © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. For research use only. Not for use in diagnostic procedures.
Editor's Notes
- Focus is on as-CNV by dPCR, which can be used for copy number analysis of specific polymorphisms in genomic or RNA sequences. Example shown in this presentation is for CYP2D6 Allele specific copy number analysis by digital PCR method Pharmacogenomics (PGx) CYP2D6 SNP and CNV analysis Digital PCR method Allele specific copy number analysis of key CYP2D6 variants
- Intro on PGx Foundational field in personalized medicine Drug development, clinical trials Individualize drug therapy selection Predict adverse reactions, dosing, response Identify increased sensitivity to drug interactions ADRs - Serious outcomes include death, hospitalization, life-threatening, disability, congenital anomaly, etc. “Streamline clinical decision making by distinguishing in advance those patients most likely to benefit from a given treatment from those who will incur cost and suffer side effects without gaining benefit” “Enhance medical product development by improving probability of success” The promise of pharmacogenomics in identifying and responding to efficacy, toxicity and ADR in commonly administered drugs is a huge opportunity that is only just beginning to have impact in the scientific community
- Transition to discussing the enzyme system and genetic variability therein that plays major role in pharmacogenetic testing. 85% of medications metabolized through a CYP450 pathway Assessing genetic variability in key enzymes is foundation of pgx testing PharmGKB – lists 52 VIP Pharmgenes involved in Phase I or Phase II ADME: absorption, distribution, metabolism, excretion (pharmacokinetics) Drug metabolism is biphasic and consists of stepwise biotransformation and synthesis reactions. Phase 1 (modification/biotransformation) consists of the oxidation (hydroxylation), hydrolysis, or reduction of a lipid-soluble or nonpolar drug. Carried out by mixed function oxidases, often in the liver. (CYP P450, Flavins, ADH, oxidases) Phase 2 (conjugation/synthesis) consists of the conjugation of a drug or its metabolite with an endogenous compound (predominantly glycine, sulfate, or glucuronic acid). Catalysed by transferases; e.g. glutathioneS-transferases (GSTs). Phase 3 – further modification and excretion By a variety of membrane transporters of the multidrug resistance protein (MRP) family. The result of either phase of metabolism is the production of metabolites that are generally more polar than the parent drug and are more readily excreted in the bile or urine. Both phases of biotransformation are the result of drug interaction with enzymes present in plasma, cytoplasm, mitochondria, and endoplasmic reticulum.
- Genotyping the CYP2D6 gene is challenging: CYP2D6 is located in a gene cluster with 2 highly homologous pseudogenes the gene is highly polymorphic: both SNP genoptying and copy number variation analysis are required there is significant variation in allele frequencies among people of different geographical origins. The entire gene can be deleted or duplicated. Several different CYP2D6 allelic variants have been reported to exist as duplications, with from 2–12 tandem copies. Gene duplication allele frequencies range from about 1 to about 30%, depending on the population. Gene conversion events refers to the fact that some CYP2D6 alleles contain some CYP2D7 seudogene sequences Several commonCYP2D6 alleles are comprised of combinations of 3 or more polymorphisms, many of which are shared by as many as 20 different alleles. Select common polymorphisms are shown in the figure. It is therefore important to genotype relatively large numbers of polymorphisms across the entire CYP2D6 gene and promoter sequence so that haplotype and genotype can be inferred. CYP2D6 genotypes can usually be unambiguously assigned on the basis of analysis of approximately 20 polymorphic sites.
- PGx Nomenclature: Star alleles are haplotypes (polymorphisms are inherited together) that are associated with enzyme function level. An activity scoring system is used to catalog the level of enzyme function. For example: *4 alleles are the most common null allele. They are defined by the 1846G>A splicing defect => no enzyme, no activity. (*4 alleles can be duplicated or found in tandem with alleles that carry pseudogene sequences) Need allele-specific copy number solution for the major duplicated CYP2D6 alleles (*1, *2, *4, *9, *10, *17, & *35). You’ll see why this is important in the next slide.
- It is the combination of star allele haplotypes, the diplotype, that predicts the DME phenotype of individual. This dme information is used to recommend appropriate drug doses. Example: CYP2D6 converts codeine to morphine, which is the effector drug. Alternates for ultra & poor include morphine & nonopiod analgesics When an individual carries a duplication allele and is heterozygous for alleles of different functional classes, it is important to know which allele was duplicated to know the phenotype of the individual. (as illustrated in the next slide) Scoring: full activity = 1.0 Reduced activity = 0.5 No activity = 0 – includes *4 splicing defect and *5 gene deletion allele
- When an individual carries a duplication allele and is heterozygous for alleles of different functional classes, it is important to know which allele was duplicated to know the phenotype of the individual. Example 1: if the individual carries 2 full function and 1reduced function allele, they are an ultrarapid metabolizer and should not use drugs metabolized by CYP2D6 that could be toxic to them. If they carry 2 reduced and 1 full funciton, they are an extensive metabolizer, they can use such drugs (age/weight/sex – specific dosing) Example 2: if the individual carries 2 reduced and 1 no function allele, they are an extensive metabolizer; but if they carry 2 no function alleles and 1 reduced they are an intermediate metabolizer and might not respond as well to CYP2D6-metabolized drugs (track their progress).
- Shown is the high throughput PGx experiment workflow: TaqMan SNP genotyping assay (DME collection) run on OA (or 384-well plates) on QS12k Flex A VIC or FAM TaqMan probe for each SNP allele analyze genotypes using TaqMan Genotyper TaqMan Copy Number assays to DME genes with CNV (CYP2D6) run in duplex rxns with reference assay on plates on QS12k analyze with CopyCaller software by ddCt (relative dCt) method. TaqMan SNP genotype and copy number experiment results can be translated to star allele diplotype results using AlleleTyper translation software. Matches genetic pattern information with a translation table containing star allele diplotype patterns Reports out the star allele diplotype call for samples ~1-2% of samples may carry duplicated alleles and be heterozygous for alleles of different functional classes. The duplicated allele cannot always be discerned by translation analsysis. Developed a new workflow for allele-specific CN analysis using SNP assays and dPCR Note: no other PGx platform provides allele-specific CN analysis
- How is that done? This is a high level schematic of a digital workflow. What’s nice is that the upfront preparation uses all of the standard techniques that are currently employed today for genomic and RNA extraction- if they work for PCR, they’ll work for digital PCR. What that also means is any samples that you might have in your freezer can be immediately analyzed by digital PCR. What’s different from running a real-time experiment versus a digital experiment is seen in the second step of this schematic. Rather than running a single reaction and driving a Ct (a measurement of fluorescense that’s created as that molecule amplifies), we instead partition that starting material in 10’s to 100’s or thousands of partitions to get to a digital range. The key to digital PCR is that not every partition (reaction) receives a molecule of interest- defined as setting up replicates at a limiting dilution. In the schematic, this is represented by partitions with DNA and some without. As seen in the third part of the schematic, any partition that receives a molecule will amplify via PCR to a level that is detectable. The amplified targets are represented in red and are called “positive “reactions. The greys do not exhibit amplification as they are empty and are known as “negative” reactions. And based on formulas that have been developed for analyzing Digital PCR, we count the number of negative reactions, apply the formula, and that will tell you how many molecules you have in your sample.
- Input concentration is 1ug input per 50uL digest volume. 1:10 dilution prior to dPCR. Final concentration in dPCR is 0.2ug/uL
- Note that *2 SNPs are found in many other alleles
- Shown are the known * allele diplotypes for these Coriell repository cell line gDNAs. The major target SNPs for the indicated * alleles listed were tested by allele-specific CN dPCR using SNP assays - to determine the allele ratios - the expected ratios of the FAM and VIC – labelled SNP assay probes are shown We will show example data for the indicated 2 and 3 –copy samples run with assays to *10 100C>T (reduced allele) and *4 1846G>A (nonfunctional allele). If anyone asks: NA17209 2 + *36 *1/*4-*36 This sample contains 3 CYP2D6 alleles. 2 are full length and 1 (*36 allele) contains gene conversion to pseudogene CYP2D7 sequences in exon 9. *36 is nonfunctional (and found at highest frequency in Asian populations) We can distinguish full length from *36 alleles using commercial TaqMan copy number assays specific for CYP2D6 exon 9 and intron 2 or intron 6 sequences. The exon 9 assay amplifies only full length alleles – not *36 alleles – whereas the intron 2 & 6 assays amplify these as well.
- Current: manual count of allele plus wells containing both alleles to get totals for each allele. Then derive the ratio for FAM to VIC For 2 copy sample: 1:1 For 3 copy sample: 1T:2C for *10 assays and 2A:1G for *4 assay In progress: algorithm that counts empties and one allele?? (what to say)
- Showing the results of duplicate runs (std dev not shown) for 5 of the assays (2 still being optimized??) Only the FAM % in each sample is shown: average vs expected 50% for 2C and 33% or 67% for 3C; 0% & 100% also tested to determine background. Actual and expected ratio %s very close Very easy to identify the duplicated allele in 3C het samples