Skip to main content

Copy number variation profiling in pharmacogenes using panel-based exome resequencing and correlation to human liver expression

Abstract

Structural variants including copy number variations (CNV) have gained widespread attention, especially in pharmacogenomics but for several genes functional relevance and clinical evidence are still lacking. Detection of CNVs in next-generation sequencing data is challenging but offers widespread applications. We developed a cohort-based CNV detection workflow to extract CNVs from read counts of targeted NGS of 340 genes involved in absorption, distribution, metabolism and excretion (ADME) of drugs. We applied our method to 150 human liver tissue samples and correlated identified CNVs to mRNA expression levels. In total, we identified 445 deletions (73%) and 167 duplications (27%) in 36 pharmacogenes including all well-known CNVs of CYPs, GSTs, SULTs, UGTs, numerous described rare CNVs of CYP2E1, SLC16A3 or UGT2B15 as well as novel observations, e.g., for SLC22A12, SLC22A17 and GPS2 (G Protein Pathway Suppressor 2). We were able to fine-map complex CNVs of CYP2A6 and CYP2D6 with exon resolution. Correlation analysis confirmed known expression patterns for common CNVs and suggested an influence on expression variability for some rare CNVs. Our straightforward CNV detection workflow can be easily applied to any NGS coverage data and helped to analyze CNVs in an ADME-NGS panel of 340 pharmacogenes to improve genotype–phenotype correlations.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Availability of data and materials

The NGS dataset used and analyzed in the current study is available from the corresponding author on reasonable request. The Illumina expression array data are available through Gene Expression Omnibus (GEO) Series accession number GSE32504.

References

  • Ahlmann-Eltze C (2019) ggsignif: Significance Brackets for ‘ggplot2’. R package version 0.5.0. https://CRAN.R-project.org/package=ggsignif

  • Bánlaki Z, Doleschall M, Rajczy K, Fust G, Szilágyi A (2012) Fine-tuned characterization of RCCX copy number variants and their relationship with extended MHC haplotypes. Genes Immun 13(7):530–535

    PubMed  Article  CAS  Google Scholar 

  • Butler MW, Hackett NR, Salit J, Strulovici-Barel Y, Omberg L, Mezey J et al (2011) Glutathione S-transferase copy number variation alters lung gene expression. Eur Respir J 38(1):15–28

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Conrad DF, Pinto D, Redon R, Feuk L, Gokcumen O, Zhang Y et al (2010) Origins and functional impact of copy number variation in the human genome. Nature 464(7289):704–712

    CAS  PubMed  Article  Google Scholar 

  • Craddock N, Hurles ME, Cardin N, Pearson RD, Plagnol V, Robson S et al (2010) Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls. Nature 464(7289):713–720

    CAS  PubMed  Article  Google Scholar 

  • Di Iulio J, Fayet A, Arab-Alameddine M, Rotger M, Lubomirov R, Cavassini M et al (2009) In vivo analysis of efavirenz metabolism in individuals with impaired CYP2A6 function. Pharmacogenet Genomics 19(4):300–309

    PubMed  Article  CAS  Google Scholar 

  • Dunnenberger HM, Crews KR, Hoffman JM, Caudle KE, Broeckel U, Howard SC et al (2015) Preemptive clinical pharmacogenetics implementation. Current programs in five US medical centers. Annu Rev Pharmacol Toxicol 55:89–106

    CAS  PubMed  Article  Google Scholar 

  • Émond J-P, Labriet A, Desjardins S, Rouleau M, Villeneuve L, Hovington H et al (2019) Factors affecting interindividual variability of hepatic UGT2B17 protein expression examined using a novel specific monoclonal antibody. Drug Metab Dispos 47(5):444–452

    PubMed  Article  CAS  Google Scholar 

  • Endrizzi K, Fischer J, Klein K, Schwab M, Nüssler A, Neuhaus P et al (2002) Discriminative quantification of cytochrome P4502D6 and 2D7/8 pseudogene expression by TaqMan real-time reverse transcriptase polymerase chain reaction. Anal Biochem 300(2):121–131

    CAS  PubMed  Article  Google Scholar 

  • Friedrichsen M, Poulsen P, Wojtaszewski J, Hansen PR, Vaag A, Rasmussen HB (2013) Carboxylesterase 1 gene duplication and mRNA expression in adipose tissue are linked to obesity and metabolic function. PLoS One 8(2):e56861

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Fukami T, Nakajima M, Maruichi T, Takahashi S, Takamiya M, Aoki Y et al (2008) Structure and characterization of human carboxylesterase 1A1, 1A2, and 1A3 genes. Pharmacogenet Genomics 18(10):911–920

    CAS  PubMed  Article  Google Scholar 

  • Gaedigk A, Twist GP, Leeder JS (2012) CYP2D6, SULT1A1 and UGT2B17 copy number variation. Quantitative detection by multiplex PCR. Pharmacogenomics 13(1):91–111

    CAS  PubMed  Article  Google Scholar 

  • Gulilat M, Lamb T, Teft WA, Wang J, Dron JS, Robinson JF et al (2019) Targeted next generation sequencing as a tool for precision medicine. BMC Med Genomics 12(1):81

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Haberl M, Anwald B, Klein K, Weil R, Fu C, Gepdiremen A et al (2005) Three haplotypes associated with CYP2A6 phenotypes in Caucasians. Pharmacogenet Genomics 15(9):609–624

    CAS  PubMed  Article  Google Scholar 

  • Hahne F, Ivanek R (2016) Visualizing genomic data using gviz and bioconductor. Methods Mol Biol 1418:335–351

    PubMed  Article  Google Scholar 

  • He Y, Hoskins JM, McLeod HL (2011) Copy number variants in pharmacogenetic genes. Trends Mol Med 17(5):244–251

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Hebbring SJ, Moyer AM, Weinshilboum RM (2008) Sulfotransferase gene copy number variation. Pharmacogenetics and function. Cytogenet Genome Res 123(1–4):205–210

    CAS  PubMed  Article  Google Scholar 

  • Henrichsen CN, Vinckenbosch N, Zöllner S, Chaignat E, Pradervand S, Schütz F et al (2009) Segmental copy number variation shapes tissue transcriptomes. Nat Genet 41(4):424–429

    CAS  PubMed  Article  Google Scholar 

  • Hoffman SM, Nelson DR, Keeney DS (2001) Organization, structure and evolution of the CYP2 gene cluster on human chromosome 19. Pharmacogenetics 11(8):687–698

    CAS  PubMed  Article  Google Scholar 

  • Hoh BP, Sam SS, Umi SH, Mahiran M, Nik Khairudin NY, Rafidah Hanim S et al (2014) A novel rare copy number variant of the ABCF1 gene identified among dengue fever patients from Peninsular Malaysia. Genet Mol Res 13(1):980–985

    CAS  PubMed  Article  Google Scholar 

  • Kassambara A (2018) ggpubr: ‘ggplot2’ Based Publication Ready Plots. R package version 0.2. https://CRAN.R-project.org/package=ggpubr

  • Kerkhof J, Schenkel LC, Reilly J, McRobbie S, Aref-Eshghi E, Stuart A et al (2017) Clinical validation of copy number variant detection from targeted next-generation sequencing panels. J Mol Diagn 19(6):905–920

    CAS  PubMed  Article  Google Scholar 

  • Kim I-W, Han N, Kim MG, Kim T, Oh JM (2015) Copy number variability analysis of pharmacogenes in patients with lymphoma, leukemia, hepatocellular, and lung carcinoma using The Cancer Genome Atlas data. Pharmacogenet Genomics 25(1):1–7

    PubMed  Article  CAS  Google Scholar 

  • Klein K, Tremmel R, Winter S, Fehr S, Battke F, Scheurenbrand T et al (2019) A new panel-based next-generation sequencing method for ADME genes reveals novel associations of common and rare variants with expression in a human liver cohort. Front Genet 10:7

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Kozyra M, Ingelman-Sundberg M, Lauschke VM (2017) Rare genetic variants in cellular transporters, metabolic enzymes, and nuclear receptors can be important determinants of interindividual differences in drug response. Genet Med 19(1):20–29

    CAS  PubMed  Article  Google Scholar 

  • Krepischi-Santos ACV, Rajan D, Temple IK, Shrubb V, Crolla JA, Huang S et al (2009) Constitutional haploinsufficiency of tumor suppressor genes in mentally retarded patients with microdeletions in 17p13.1. Cytogenet Genome Res 125(1):1–7

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Kuroda Y, Ohashi I, Tominaga M, Saito T, Nagai J-I, Ida K et al (2014) De novo duplication in a patient with intellectual disability and obesity. Am J Med Genet A 164A(6):1550–1554

    PubMed  Article  CAS  Google Scholar 

  • Lauschke VM, Ingelman-Sundberg M (2019) Prediction of drug response and adverse drug reactions. From twin studies to next generation sequencing. Eur J Pharm Sci 130:65–77

    CAS  PubMed  Article  Google Scholar 

  • Li H, Durbin R (2010) Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26(5):589–595

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Ma I, Allan AL (2011) The role of human aldehyde dehydrogenase in normal and cancer stem cells. Stem Cell Rev 7(2):292–306

    CAS  Article  Google Scholar 

  • Martis S, Mei H, Vijzelaar R, Edelmann L, Desnick RJ, Scott SA (2013) Multi-ethnic cytochrome-P450 copy number profiling. Novel pharmacogenetic alleles and mechanism of copy number variation formation. Pharmacogenomics J 13(6):558–566

    CAS  PubMed  Article  Google Scholar 

  • Ménard V, Eap O, Harvey M, Guillemette C, Lévesque E (2009) Copy-number variations (CNVs) of the human sex steroid metabolizing genes UGT2B17 and UGT2B28 and their associations with a UGT2B15 functional polymorphism. Hum Mutat 30(9):1310–1319

    PubMed  Article  CAS  Google Scholar 

  • Meyer UA (2004) Pharmacogenetics–five decades of therapeutic lessons from genetic diversity. Nat Rev Genet 5(9):669–676

    CAS  PubMed  Article  Google Scholar 

  • Meyer UA, Zanger UM, Schwab M (2013) Omics and drug response. Annu Rev Pharmacol Toxicol 53:475–502

    CAS  PubMed  Article  Google Scholar 

  • Nord AS, Lee M, King M-C, Walsh T (2011) Accurate and exact CNV identification from targeted high-throughput sequence data. BMC Genomics 12:184

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Numanagić I, Malikić S, Ford M, Qin X, Toji L, Radovich M et al (2018) Allelic decomposition and exact genotyping of highly polymorphic and structurally variant genes. Nat Commun 9(1):828

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Oliveira C, Wolf T (2019) CNVPanelizer. Reliable CNV detection in targeted sequencing applications. R package version 1.4.0. https://bioconductor.org/packages/release/bioc/html/CNVPanelizer.html

  • Parajes S, Quinteiro C, Domínguez F, Loidi L (2008) High frequency of copy number variations and sequence variants at CYP21A2 locus. Implication for the genetic diagnosis of 21-hydroxylase deficiency. PLoS One 3(5):e2138

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Pirmohamed M (2014) Personalized pharmacogenomics. Predicting efficacy and adverse drug reactions. Annu Rev Genomics Hum Genet 15:349–370

    CAS  PubMed  Article  Google Scholar 

  • Plagnol V, Curtis J, Epstein M, Mok KY, Stebbings E, Grigoriadou S et al (2012) A robust model for read count data in exome sequencing experiments and implications for copy number variant calling. Bioinformatics 28(21):2747–2754

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Povysil G, Tzika A, Vogt J, Haunschmid V, Messiaen L, Zschocke J et al (2017) panelcn.MOPS. Copy-number detection in targeted NGS panel data for clinical diagnostics. Hum Mutat 38(7):889–897

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Relling MV, Evans WE (2015) Pharmacogenomics in the clinic. Nature 526(7573):343–350

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Rose-Zerilli MJ, Barton SJ, Henderson AJ, Shaheen SO, Holloway JW (2009) Copy-number variation genotyping of GSTT1 and GSTM1 gene deletions by real-time PCR. Clin Chem 55(9):1680–1685

    CAS  PubMed  Article  Google Scholar 

  • Ruderfer DM, Hamamsy T, Lek M, Karczewski KJ, Kavanagh D, Samocha KE et al (2016) Patterns of genic intolerance of rare copy number variation in 59,898 human exomes. Nat Genet 48(10):1107–1111

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Santos M, Niemi M, Hiratsuka M, Kumondai M, Ingelman-Sundberg M, Lauschke VM et al (2018) Novel copy-number variations in pharmacogenes contribute to interindividual differences in drug pharmacokinetics. Genet Med 20(6):622–629

    CAS  PubMed  Article  Google Scholar 

  • Schaeffeler E, Schwab M, Eichelbaum M, Zanger UM (2003) CYP2D6 genotyping strategy based on gene copy number determination by TaqMan real-time PCR. Hum Mutat 22(6):476–485

    CAS  PubMed  Article  Google Scholar 

  • Schärfe CPI, Tremmel R, Schwab M, Kohlbacher O, Marks DS (2017) Genetic variation in human drug-related genes. Genome Med 9(1):117

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Schröder A, Klein K, Winter S, Schwab M, Bonin M, Zell A et al (2013) Genomics of ADME gene expression. Mapping expression quantitative trait loci relevant for absorption, distribution, metabolism and excretion of drugs in human liver. Pharmacogenomics J 13(1):12–20

    PubMed  Article  CAS  Google Scholar 

  • Slowikowski K (2018) ggrepel: Automatically Position Non-Overlapping Text Labels with ‘ggplot2’. R package version 0.8.0. https://CRAN.R-project.org/package=ggrepel

  • Sprenger R, Schlagenhaufer R, Kerb R, Bruhn C, Brockmöller J, Roots I et al (2000) Characterization of the glutathione S-transferase GSTT1 deletion. Discrimination of all genotypes by polymerase chain reaction indicates a trimodular genotype-phenotype correlation. Pharmacogenetics 10(6):557–565

    CAS  PubMed  Article  Google Scholar 

  • Tremmel R, Klein K, Winter S, Schaeffeler E, Zanger UM (2016) Gene copy number variation analysis reveals dosage-insensitive expression of CYP2E1. Pharmacogenomics J 16(6):551–558

    CAS  PubMed  Article  Google Scholar 

  • Tremmel R, Herrmann K, Engst W, Meinl W, Klein K, Glatt H et al (2017) Methyleugenol DNA adducts in human liver are associated with SULT1A1 copy number variations and expression levels. Arch Toxicol 91(10):3329–3339

    CAS  PubMed  Article  Google Scholar 

  • van der Wouden CH, Cambon-Thomsen A, Cecchin E, Cheung KC, Dávila-Fajardo CL, Deneer VH et al (2017) Implementing pharmacogenomics in Europe. Design and implementation strategy of the ubiquitous pharmacogenomics consortium. Clin Pharmacol Ther 101(3):341–358

    PubMed  Article  Google Scholar 

  • Wickham H (2017) tidyverse: Easily Install and Load the ‘Tidyverse’. R package version 1.2.1. https://CRAN.R-project.org/package=tidyverse

  • Woodwark C, Bateman A (2011) The characterisation of three types of genes that overlie copy number variable regions. PLoS One 6(5):e14814

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Xu S, Wang Y, Roe B, Pearson WR (1998) Characterization of the human class Mu glutathione S-transferase gene cluster and the GSTM1 deletion. J Biol Chem 273(6):3517–3527

    CAS  PubMed  Article  Google Scholar 

  • Yang W, Zhang Y, Fu F, Li R (2013) High-resolution array-comparative genomic hybridization profiling reveals 20q13.33 alterations associated with ovarian endometriosis. Gynecol Endocrinol 29(6):603–607

    CAS  PubMed  Article  Google Scholar 

  • Zanger UM, Schwab M (2013) Cytochrome P450 enzymes in drug metabolism. Regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol Ther 138(1):103–141

    CAS  PubMed  Article  Google Scholar 

  • Zhou J, Lemos B, Dopman EB, Hartl DL (2011) Copy-number variation. The balance between gene dosage and expression in Drosophila melanogaster. Genome Biol Evol 3:1014–1024

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

We thank Dr. Florian Büttner for statistical expertise and discussions. The excellent technical assistance of Igor Liebermann is gratefully acknowledged. This study was supported by the Robert Bosch Foundation, Stuttgart, Germany and the European Commission Horizon 2020-PHC-2015 Grant U-PGx 668353.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Roman Tremmel.

Ethics declarations

Conflict of interest

SF, FB, TS, and SB were employed by CeGaT GmbH, Tübingen. The remaining authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tremmel, R., Klein, K., Battke, F. et al. Copy number variation profiling in pharmacogenes using panel-based exome resequencing and correlation to human liver expression. Hum Genet 139, 137–149 (2020). https://doi.org/10.1007/s00439-019-02093-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00439-019-02093-7