Advertisement

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

  • Roman TremmelEmail author
  • Kathrin Klein
  • Florian Battke
  • Sarah Fehr
  • Stefan Winter
  • Tim Scheurenbrand
  • Elke Schaeffeler
  • Saskia Biskup
  • Matthias Schwab
  • Ulrich M. Zanger
Original Investigation
  • 82 Downloads

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.

Notes

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.

Compliance with ethical standards

Conflict of interest

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

Supplementary material

439_2019_2093_MOESM1_ESM.docx (1.2 mb)
Supplementary material 1 (DOCX 1226 kb)
439_2019_2093_MOESM2_ESM.pdf (190 kb)
Supplementary material 2 (PDF 189 kb)
439_2019_2093_MOESM3_ESM.pdf (285 kb)
Supplementary material 3 (PDF 284 kb)
439_2019_2093_MOESM4_ESM.pdf (3.2 mb)
Supplementary material 4 (PDF 3309 kb)
439_2019_2093_MOESM5_ESM.pdf (1.8 mb)
Supplementary material 5 (PDF 1837 kb)
439_2019_2093_MOESM6_ESM.pdf (359 kb)
Supplementary material 6 (PDF 358 kb)

References

  1. Ahlmann-Eltze C (2019) ggsignif: Significance Brackets for ‘ggplot2’. R package version 0.5.0. https://CRAN.R-project.org/package=ggsignif
  2. 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):530–535PubMedCrossRefGoogle Scholar
  3. 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):15–28PubMedPubMedCentralCrossRefGoogle Scholar
  4. 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):704–712PubMedCrossRefGoogle Scholar
  5. 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):713–720PubMedCrossRefGoogle Scholar
  6. 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):300–309PubMedCrossRefGoogle Scholar
  7. 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 55:89–106PubMedCrossRefGoogle Scholar
  8. 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):444–452PubMedCrossRefGoogle Scholar
  9. 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):121–131PubMedCrossRefGoogle Scholar
  10. 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):e56861PubMedPubMedCentralCrossRefGoogle Scholar
  11. 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):911–920PubMedCrossRefGoogle Scholar
  12. Gaedigk A, Twist GP, Leeder JS (2012) CYP2D6, SULT1A1 and UGT2B17 copy number variation. Quantitative detection by multiplex PCR. Pharmacogenomics 13(1):91–111PubMedCrossRefGoogle Scholar
  13. 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):81PubMedPubMedCentralCrossRefGoogle Scholar
  14. 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):609–624PubMedCrossRefGoogle Scholar
  15. Hahne F, Ivanek R (2016) Visualizing genomic data using gviz and bioconductor. Methods Mol Biol 1418:335–351PubMedCrossRefGoogle Scholar
  16. He Y, Hoskins JM, McLeod HL (2011) Copy number variants in pharmacogenetic genes. Trends Mol Med 17(5):244–251PubMedPubMedCentralCrossRefGoogle Scholar
  17. Hebbring SJ, Moyer AM, Weinshilboum RM (2008) Sulfotransferase gene copy number variation. Pharmacogenetics and function. Cytogenet Genome Res 123(1–4):205–210PubMedCrossRefGoogle Scholar
  18. 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):424–429PubMedCrossRefGoogle Scholar
  19. Hoffman SM, Nelson DR, Keeney DS (2001) Organization, structure and evolution of the CYP2 gene cluster on human chromosome 19. Pharmacogenetics 11(8):687–698PubMedCrossRefGoogle Scholar
  20. 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):980–985PubMedCrossRefGoogle Scholar
  21. Kassambara A (2018) ggpubr: ‘ggplot2’ Based Publication Ready Plots. R package version 0.2. https://CRAN.R-project.org/package=ggpubr
  22. 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):905–920PubMedCrossRefGoogle Scholar
  23. 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):1–7PubMedCrossRefGoogle Scholar
  24. 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 10:7PubMedPubMedCentralCrossRefGoogle Scholar
  25. 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):20–29PubMedCrossRefGoogle Scholar
  26. 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):1–7PubMedPubMedCentralCrossRefGoogle Scholar
  27. 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):1550–1554PubMedCrossRefGoogle Scholar
  28. 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 130:65–77PubMedCrossRefGoogle Scholar
  29. Li H, Durbin R (2010) Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26(5):589–595PubMedPubMedCentralCrossRefGoogle Scholar
  30. Ma I, Allan AL (2011) The role of human aldehyde dehydrogenase in normal and cancer stem cells. Stem Cell Rev 7(2):292–306CrossRefGoogle Scholar
  31. 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):558–566PubMedCrossRefGoogle Scholar
  32. 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):1310–1319PubMedCrossRefGoogle Scholar
  33. Meyer UA (2004) Pharmacogenetics–five decades of therapeutic lessons from genetic diversity. Nat Rev Genet 5(9):669–676PubMedCrossRefGoogle Scholar
  34. Meyer UA, Zanger UM, Schwab M (2013) Omics and drug response. Annu Rev Pharmacol Toxicol 53:475–502PubMedCrossRefGoogle Scholar
  35. Nord AS, Lee M, King M-C, Walsh T (2011) Accurate and exact CNV identification from targeted high-throughput sequence data. BMC Genomics 12:184PubMedPubMedCentralCrossRefGoogle Scholar
  36. 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):828PubMedPubMedCentralCrossRefGoogle Scholar
  37. 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
  38. 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):e2138PubMedPubMedCentralCrossRefGoogle Scholar
  39. Pirmohamed M (2014) Personalized pharmacogenomics. Predicting efficacy and adverse drug reactions. Annu Rev Genomics Hum Genet 15:349–370PubMedCrossRefGoogle Scholar
  40. 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):2747–2754PubMedPubMedCentralCrossRefGoogle Scholar
  41. 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):889–897PubMedPubMedCentralCrossRefGoogle Scholar
  42. Relling MV, Evans WE (2015) Pharmacogenomics in the clinic. Nature 526(7573):343–350PubMedPubMedCentralCrossRefGoogle Scholar
  43. 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):1680–1685PubMedCrossRefGoogle Scholar
  44. 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):1107–1111PubMedPubMedCentralCrossRefGoogle Scholar
  45. 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):622–629PubMedCrossRefGoogle Scholar
  46. 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):476–485PubMedCrossRefGoogle Scholar
  47. Schärfe CPI, Tremmel R, Schwab M, Kohlbacher O, Marks DS (2017) Genetic variation in human drug-related genes. Genome Med 9(1):117PubMedPubMedCentralCrossRefGoogle Scholar
  48. 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):12–20PubMedCrossRefGoogle Scholar
  49. 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
  50. 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):557–565PubMedCrossRefGoogle Scholar
  51. 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):551–558PubMedCrossRefGoogle Scholar
  52. 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):3329–3339PubMedCrossRefGoogle Scholar
  53. 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):341–358PubMedCrossRefGoogle Scholar
  54. Wickham H (2017) tidyverse: Easily Install and Load the ‘Tidyverse’. R package version 1.2.1. https://CRAN.R-project.org/package=tidyverse
  55. Woodwark C, Bateman A (2011) The characterisation of three types of genes that overlie copy number variable regions. PLoS One 6(5):e14814PubMedPubMedCentralCrossRefGoogle Scholar
  56. 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):3517–3527PubMedCrossRefGoogle Scholar
  57. 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):603–607PubMedCrossRefGoogle Scholar
  58. 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):103–141PubMedCrossRefPubMedCentralGoogle Scholar
  59. 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 3:1014–1024PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Roman Tremmel
    • 1
    • 2
    Email author
  • Kathrin Klein
    • 1
    • 2
  • Florian Battke
    • 3
    • 4
  • Sarah Fehr
    • 3
    • 4
  • Stefan Winter
    • 1
    • 2
  • Tim Scheurenbrand
    • 3
    • 4
  • Elke Schaeffeler
    • 1
    • 2
    • 5
  • Saskia Biskup
    • 3
    • 4
  • Matthias Schwab
    • 1
    • 5
    • 6
  • Ulrich M. Zanger
    • 1
    • 2
  1. 1.Dr. Margarete Fischer-Bosch-Institute of Clinical PharmacologyStuttgartGermany
  2. 2.University of TübingenTübingenGermany
  3. 3.CeGaT GmbHTübingenGermany
  4. 4.Praxis für Humangenetik TübingenTübingenGermany
  5. 5.iFIT Cluster of Excellence, University of TübingenTübingenGermany
  6. 6.Departments of Clinical Pharmacology, Pharmacy and BiochemistryUniversity of TübingenTübingenGermany

Personalised recommendations