Application Note. *For Research Use ... and biotech companies need effective tools to investigate ... software* (version 3.1.50.00) and iPLEX Gold* biochemistry ...
Application Note Development and Research Validation of the iPLEX® ADME PGx Panel on the MassARRAY® System Robin E. Everts, Ph.D., Heath Metzler, Vivian Huang Ph.D., Christiane Honisch Ph.D., Rene Nunez Sequenom, Inc., 3595 John Hopkins Ct, San Diego, CA 92121
Introduction The goals of pharmacogenetic research are to understand and validate drug targets and metabolic pathways, identify optimal dosage, improve drug safety, and understand adverse side effects. Meeting these goals will aid in making safe and effective drugs for individuals for whom the drugs are prescribed. For these reasons, pharmaceutical and biotech companies need effective tools to investigate biomarkers associated with drug absorption, distribution, metabolism and excretion (ADME). Over 169 genes are known to be involved in drug transport and/or metabolism (1). For these genes, the PharmaADME working group (2) has identified a set of approximately 180 assays that are considered to be the core set of mutations that influence most of the known drug metabolism and transport issues. A powerful tool for investigating and screening associations to drug response A new ADME pharmacogenomics panel launched by Sequenom, covers >99% of the PharmaADME working group core list. The iPLEX ADME PGx panel* (Fig 1) was developed for quick and easy screening of known, high value target genes associated with drug metabolism and toxicity. The iPLEX ADME PGx panel screens for 192 polymorphisms (SNP, INDEL, and CNV) in 36 genes and 200 assays using Typer Assay Designer software* (version 3.1.50.00) and iPLEX Gold* biochemistry (Table 1). The panel provides flexible options for analyzing a small or large sample size while keeping reagent costs down. This application note describes the results of the of iPLEX ADME PGx panel at four different sites using two independent sets of DNA samples (HapMap samples and clinical samples) on the MassARRAY System*.
Figure 1. iPLEX ADME PGx panel
Table 1. Distribution of the 200 assays that represent the Sequenom iPLEX ADME PGx panel Gene
SNP
ABCB1
4
GSTT1
1
ABCC2
6
GSTT2b
1
ABCG2
2
NAT1
7
COMT
3
NAT2
8
CYP1A1
7
SLC15A2
4
CYP1A2
4
SLC22A1
14
CYP2A6
9
2
SLC22A2
5
CYP2B6
5
2
SLC22A6
1
CYP2C19
10
SLCO1B1
9
CYP2C8
6
SLCO1B3
2
CYP2C9
13
SLCO2B1
1
CYP2D6
24
SULT1A1
3
CYP2E1
2
TPMT
5
CYP3A4
4
UGT1A1
5
CYP3A5
5
UGT2B15
1
DPYD
6
GSTM1
1
GSTP1
2
CNV
Conversion
2
3
1
Gene
SNP
UGT2B17 1
CNV
1
2
UGT2B7
2
VKORC1
4
Note: 200 assays include 184 SNPs, 4 gene conversions, and 12 copy number variations. Some of the copy number variations are being interrogated by more than one assay resulting in 192 unique polymorphisms. Conversion assays interrogate the gene conversion for CYP2A6/A7 and CYP2D6/D7 intron 1.
*For Research Use Only. Not for Use in Diagnostic Procedures.
Materials and Methods Samples: Aliquots of 48 DNA samples previously genotyped at Sequenom were sent out to 4 test sites, together with all reagents and the ADME PGx software* to perform on-site testing. In addition, test sites were able to test up to 48 of their own samples and also send aliquots of the DNA to Sequenom for concordance analysis. Assays: PCR primers and Extension primers for 200 assays (184 SNP and INDEL assays, 12 CNV assays, and 4 gene conversion assays) were mixed in eight unique pools and used for amplification of target regions and subsequent interrogation of the specific base composition at the target site using single base extension with the iPLEX Gold biochemistry. Procedures: Test sites were trained using HapMap DNA samples. After successful completion of the training, test sites genotyped the same HapMap DNA plate as well as a DNA plate of their own clinical samples. At Sequenom, two independent technical personnel genotyped the samples provided by the customer. The iPLEX ADME PGx panel uses standard iPLEX Gold procedures for PCR, SAP and extension reactions (see Figure 2).
Data analysis: Data for each genotyping experiment was collected before and after manual analysis. Known genotyping data from beta site samples (HapMap DNA) was compared for concordance against data generated on the iPLEX ADME PGx panel. For customer samples, data generated at the customer site and at two Sequenom in-house sites was compared and analyzed for call rate and concordance rate. Haplotype calling: Using publicly available data for 33 genes, a lookup table was created and an Rscript written to correlate the genotypes and haplotypes or mutations, whichever is used for the genes of interest. Where possible, haplotype data from the script was compared to available haplotype data derived from other sources. The Typer Analyzer software* (version 4.0.20), is Sequenom’s proprietary software used to score and qualify polymorphisms and to create a haplotype report. Results Call rate and concordance. The iPLEX ADME PGx panel consists of 184 SNP/INDEL assays and 16 CNV/gene conversion assays. The call rate and concordance analysis were performed on all calls made. The output is summarized with respect to call rate and concordance rate between the different sites (Tables 2). The call rates were on average 97.5% for the HapMap samples, and 97.3% for each site’s clinical samples. Average concordance between the different HapMap data sets was 99.7% using HapMap data only. Based on all data points for the 48 HapMap samples (9,600 genotypes) the average concordance was 99.7%. For the clinical samples (n=207), the average concordance between the 3 sites was 99.6%. For the copy number assays, all assays were very clear on the distinction between 0 and non-0 copy numbers. Concordance for these assays was >98% with literature data (3-6).
Figure 2. iPLEX ADME PGx panel Workflow WEB SITE WWW.SEQUENOM.COM ASIA/PACIFIC +61 7 3845 3683
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Table 2. Summary data of the beta test data for Hapmap and clinical samples Call Rate* Concordance* HapMap
97.5%
99.7%
Clinical samples
97.3%
99.6%
Table 3. List of 268 Haplotypes in the iPLEX ADME PGx panel
*based on 4 independent beta sites and 2 Sequenom sites Haplotype data. Using the Admet report function in Typer software* (Figure 3), genotypes were converted to haplotypes and copy number variants (CNV) and the output report provides five tables and one figure. An example of the output of Long Table II is presented (Figure 4). For customer samples where data from other platforms was available, our haplotype results matched the previous outcome. Table 6 lists all the genes and associated haplotypes available in the iPLEX ADME PGx panel.
.
Figure 3. Admet report function in Typer
Note: For some genes that have no haplotype information, standard nomenclature based on amino acid changes were listed. Figure 4. Output of the Admet report function in Typer.
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Discussion The iPLEX ADME PGx panel containing 192 polymorphisms was successfully developed and validated on the MassARRAY system. The panel shows excellent call rate (>97%) and concordance (>99.5%) with published data. No difference in performance for the panel was detected between clinical samples and HapMap samples. The panel covers 184 SNPs, eight gene copy numbers and 4 gene conversion assays, representing over 99% of the Pharma ADME core list. Currently, for the CNV assays, distinctions between 0, and non-0 copies of a gene can be made. For CYP2D6, CYP2A6, and SULT1A1 we used segments of the genes selected earlier (7-9). For CYP2B6, GSTM1, GSTT2b, and UGT2B15 we identified new regions of homology to use for CNV analysis. This approach entails the selection of a region in the gene of interest that is present at exactly one more location in the genome that serves as the reference gene (Figure 5). This reference gene is assumed to be free of deletion
and/or duplication events and can therefore serve as a baseline competitor for quantification. For a single base extension (SBE) assay to work, amplification products need to be equal in size and there needs to be at least one base mismatch between the reference gene and gene of interest that is located in between the primer binding sites. As a result, a primer extension reaction of both targets will produce two alleles of different masses that can be visualized using MassARRAY system. The ratio of the areas of the peaks can then be calculated and the zygosity of the gene of interest can be determined. For example, if the peak for CYP2D6 is 50% of the peak height of the reference gene, a CYP2D6*5 deletion allele is present. Furthermore, if the peak for CYP2D6 is 50% higher than the reference peak, a duplication event has taken place. For GSTT1 we designed an assay using Genbank sequences AF240785 and the most common 1460 bp deletion assay (10).
Figure 5. The principle of the paralogous assay approach to genotype copy numbers of certain genes.
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One primer set (P1 and P2) amplifies two almost identical PCR fragments from two different regions in the genome. One region is the “gene of interest” (I), the other region or “reference” (R) is a different region and assumed to be free of copy number variations. Single base extension assays are then run on the minor differences between R and I contained within the amplified paralogous regions.
Figure 6. Actual data output for CNV assays using the Admet reporter function in Typer. Summary In this study, the iPLEX ADME PGx panel was evaluated at multi-centers on the MassARRAY system. The panel shows excellent call rate and concordance with published data, and no difference in performance characteristics were detected between each site’s clinical samples and HapMap samples. In conclusion, iPLEX ADME PGx panel provides a high-throughput screening method for rapid screening of known high value target genes associated with drug metabolism and toxicity. In addition, with iPLEX ADME PGx panel, researchers can distinguish the difference between zero and non-zero copy number variants, which is unique to iPLEX ADME PGx method versus other ADME profiling approaches. Reference List 1. Daly, T. M., Dumaual, C. M., Miao, X., Farmen, M. W., Njau, R. K., Fu, D. J., Bauer, N. L., Close, S., Watanabe, N., Bruckner, C. et al. (2007) Clin. Chem. 53, 1222-1230. 2. Williams, J. A., Andersson, T., Andersson, T. B., Blanchard, R., Behm, M. O., Cohen, N., Edeki, T., Franc, M., Hillgren, K. M., Johnson, K. J. et al. (2008) J. Clin. Pharmacol. 48, 849889. 3. Ramamoorthy, A., Flockhart, D. A., Hosono, N., Kubo, M., Nakamura, Y., & Skaar, T. C. (2010) Pharmacogenet. Genomics 20, 451454. 4. Xue, Y., Sun, D., Daly, A., Yang, F., Zhou, X., Zhao, M., Huang, N., Zerjal, T., Lee, C., Carter, N. P. et al. (2008) Am. J. Hum. Genet. 83, 337-346. WEB SITE WWW.SEQUENOM.COM ASIA/PACIFIC +61 7 3845 3683
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5. Zhao, Y., Marotta, M., Eichler, E. E., Eng, C., & Tanaka, H. (2009) PLoS. Genet. 5, e1000472. 6. McCarroll, S. A., Hadnott, T. N., Perry, G. H., Sabeti, P. C., Zody, M. C., Barrett, J. C., Dallaire, S., Gabriel, S. B., Lee, C., Daly, M. J. et al. (2006) Nat. Genet. 38, 86-92. 7. Söderbäck, E., Zackrisson, A. L., Lindblom, B., & Alderborn, A. (2005) Clin. Chem. 51, 522531. 8. Oscarson, M., McLellan, R. A., Gullsten, H., Yue, Q. Y., Lang, M. A., Bernal, M. L., Sinues, B., Hirvonen, A., Raunio, H., Pelkonen, O. et al. (1999) FEBS Lett. 448, 105-110. 9. Hebbring, S. J., Adjei, A. A., Baer, J. L., Jenkins, G. D., Zhang, J., Cunningham, J. M., Schaid, D. J., Weinshilboum, R. M., & Thibodeau, S. N. (2007) Hum. Mol. Genet. 16, 463-470. 10. Sprenger, R., Schlagenhaufer, R., Kerb, R., Bruhn, C., Brockmoller, J., Roots, I., & Brinkmann, U. (2000) Pharmacogenetics 10, 557-565. ®
The MassARRAY system, iPLEX ADME PGx Panel, iPLEX Gold and Typer Assay Designer software Typer Analyzer software are For Research Use Only. Not for use in Diagnostic Procedures. ® 2012 Sequenom, Inc. All rights reserved. Sequenom, iPLEX, MassARRAY, and SpectroCHIP are registered trademarks of Sequenom, Inc. Products and/or processes are covered by one or more claims of United States Patent Nos.: 6,569,385; 6,500,621; 6,300,076; 6,258,538; 7,419,787; 7,390,672; and foreign equivalents. Other U.S. and foreign patents pending. The SABER method is covered by United States Patent No.: 7,709,262 and pending application 12/496,390 which is exclusively licensed to Sequenom, Inc.
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