1The 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

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• 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

What Is Pharmacogenomics?

Figure from www.pharmainfo.net/reviews/role-pharmacogenomics-drug-development.

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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

PGx Drug Metabolism Enzymes (DMEs)

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Genotyping Highly Polymorphic CYP2D6 Is Challenging

Figure adapted from Nature Reviews Drug Discovery (2004)

• > 80 known CYP2D6 polymorphisms within coding and regulatory regions• includes SNPs, InDels, CNVs, and gene conversion events

• > 100 characterized CYP2D6 ‘star’ alleles that can contain multiple polymorphisms• Genotype analysis requires both SNP genoptyping and CNV analysis

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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 SNPsEnzyme 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

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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

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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

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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

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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

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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

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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

TaqMan® Drug Metabolism Genotyping Assays tested

VIC®

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Test Coriell gDNA Samples of Known Genotypes(2 or 3 copies of CYP2D6)

Samples Copy Number Genotype Target SNPsExpected

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 1:02850C>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

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*10 100C>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

GG

empty empty

emptyempty

T/CT/C

A/GA/G

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Accurate Calling of Copy Number Ratios

NA17116 NA17105 NA17209 NA17117 NA17116 NA17209 NA17105 NA17117

FAM % Aver-age

49.20515 64.68775 64.1713 65.28695 50.5633 33.29005 33.2844 0.36285

FAM % Ex-pected

50 66.66 66.66 66.66 50 33.33 33.33 0

5

25

45

65

FA

M % 1 of 2

1 of 3

1 of 2

1 of 3

0 of 3

2 of 3

*4 1846G>A2 of 3 2 of 3

NA17116 NA17105 NA17155 NA10859 NA17209 NA17155 NA17116 NA17155

FAM % Aver-age

48.4845 66.0888 0.49255 47.4216 53.8026 99.46075 51.0802 33.39455

FAM % Ex-pected

50 66.66 0 50 50 100 50 33.33

10

50

90

FA

M %

1 of 31 of 2

0 of 3

2 of 31 of 2

1 of 21 of 2

*17 1023C>T 3 of 3

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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

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Acknowlegements

R&DSunali PatelKathleen HayashibaraTrish HegerichDavid KeysKamini Varma

Product ManagementIain RussellElliot SheltonPaco Cifuentes

Thermo Fisher Scientific

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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.