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A Genome-Wide Approach to Identify Genetic Variants That Contribute to Etoposide-Induced Cytotoxicity

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A Genome-Wide Approach to Identify Genetic Variants That Contribute to Etoposide-Induced Cytotoxicity A genome-wide approach to identify genetic variants that contribute to etoposide-induced cytotoxicity R. Stephanie Huang*, Shiwei Duan*, Wasim K. Bleibel*, Emily O. Kistner†, Wei Zhang*, Tyson A. Clark‡, Tina X. Chen‡, Anthony C. Schweitzer‡, John E. Blume‡, Nancy J. Cox§, and M. Eileen Dolan*¶ *Section of Hematology–Oncology, †Biostatistics Consulting Laboratory, Department of Health Studies, and §Section of Genetic Medicine, Department of Medicine, University of Chicago, Chicago, IL 60637; and ‡Expression Research, Affymetrix Laboratory, Affymetrix, Inc., Santa Clara, CA 95051 Communicated by Janet D. Rowley, University of Chicago Medical Center, Chicago, IL, April 26, 2007 (received for review December 4, 2006) Large interindividual variance has been observed in sensitivity to 100 drugs. To comprehensively decipher the genetic contribution to 75 these variations in drug susceptibility, we present a genome-wide 50 % surv ival model using human lymphoblastoid cell lines from the Interna- 25 012 tional HapMap consortium, of which extensive genotypic infor- Dgur conce rtn a ti(on µµµM) SNPs mRNA expressino Phenotype mation is available, to identify genetic variants that contribute to DgCru I 50 chemotherapeutic agent-induced cytotoxicity. Our model inte- grated genotype, gene expression, and sensitivity of HapMap cell Wghole enome asso citaio n lines to drugs. Cell lines derived from 30 trios of European descent SNPs associaiuted w tgh rd IC (P<1x10 4- ) (Center d’Etude du Polymorphisme Humain population) and 30 50 trios of African descent (Yoruban population) were used. Cell Ggsenotype and eine expres on growth inhibition at increasing concentrations of etoposide for associta ino 72 h was determined by using alamarBlue assay. Gene expression SiNPs c sg/ t ran r-s e lu ta ed gene on 176 HapMap cell lines (87 Center d’Etude du Polymorphisme expressioin ( Bo refn or n c0orrected P< .05) Humain population and 89 Yoruban population) was determined by using the Affymetrix GeneChip Human Exon 1.0ST Array. We Giene express ogCn and dru I 50 cor er tal ion evaluated associations between genotype and cytotoxicity, geno- Gene expressiwoiuns c etalerro d tgh rd type and gene expression and correlated gene expression of the IC50 (P<0.05) identified candidates with cytotoxicity. The analysis identified 63 genetic variants that contribute to etoposide-induced toxicity Fig. 1. Genome-wide model to identify genetic variants important in drug cytotoxicity via expression levels. Model includes whole genome association through their effect on gene expression. These include genes that between SNP genotype and gene expression, association between genotype may play a role in cancer (AGPAT2, IL1B, and WNT5B) and genes not and gene expression and linear regression between gene expression and yet known to be associated with sensitivity to etoposide. This etoposide IC50. The image to display SNPs is courtesy of the U.S. Department unbiased method can be used to elucidate genetic variants con- of Energy’s Joint Genome Institute (Walnut Creek, CA; www.jgi.doe.gov). tributing to a wide range of cellular phenotypes induced by chemotherapeutic agents. chemotherapeutic-induced toxicity in patients, to identify those ‘‘at HapMap ͉ pharmacogenomics ͉ toxicity ͉ whole-genome association risk’’ for adverse events associated with these agents. Etoposide, a topoisomerase II inhibitor (2), was chosen to andidate gene and genome-wide approaches have been used to illustrate the utility of our model because of its wide usage in the Cidentify genes important in cellular sensitivity to drugs. Al- treatment of disseminated testicular carcinomas, lung cancer, ger- though candidate gene approaches have had reasonable success in minal malignancies, non-Hodgkin’s lymphoma, acute myelogenous identifying genes important in the mechanisms of action of drugs, leukemia, and Kaposi’s sarcoma. Etoposide is associated with bone the multigenic nature of the drug effect has limited the ability of marrow suppression, fatigue, skin rash, and diarrhea and can cause these approaches to explain much of the interindividual variation in a severe delayed toxicity, treatment-related acute myeloid leukemia drug effect. Genome-wide approaches open up the possibility to or myelodysplastic syndrome (3, 4). Treatment-induced toxicity has identify multiple components or pathways that contribute to cell hindered the use of this agent to its full potential. Therefore, the susceptibility to drugs. It is particularly challenging to study genes that contribute to cellular sensitivity to chemotherapeutic drugs, because their antitumor effect is dictated by somatic mutation in the Author contributions: R.S.H. and S.D. contributed equally to this work; R.S.H., S.D., and M.E.D. designed research; R.S.H., S.D., W.K.B., T.A.C., and T.X.C. performed research; T.A.C. tumor and toxic effects controlled by host genomic effects. Fur- and J.E.B. contributed new reagents/analytic tools; R.S.H., S.D., E.O.K., W.Z., A.C.S., N.J.C., thermore, chemotherapy cannot be given to noncancerous family and M.E.D. analyzed data; and R.S.H. and M.E.D. wrote the paper. members for classical genetic studies. Recently, the International Conflict of interest statement: T.A.C., T.X.C., A.C.S., and J.E.B. are employees of Affymetrix, HapMap Consortium genotyped cell lines derived from trios of Inc., 3420 Central Expressway, Santa Clara, CA 95051. European and Yoruban descent, providing an extremely rich data Abbreviations: LCLs, lymphoblastoid cell lines; QTDT, quantitative transmission disequilib- set for genotype–drug effect correlations (1). rium test; GO, gene ontology; LD, linkage disequilibrium; CEU, Center d’Etude du Poly- Using data generated on the HapMap cell lines, we designed a morphisme Humain population; YRI, Yoruban population. three-way model, correlating genotype, gene expression, and cyto- Data deposition: The expression data reported in this paper have been deposited in the National Center for Biotechnology Information Gene Omnibus database (GEO accession toxicity data, with the aim of identifying potentially functional SNPs no. GSE 7792), and the phenotype data have been deposited in the PharmGKB database, and/or haplotypes associated with chemotherapeutic agent-induced www.pharmgkb.org (accession no. PS206922). cytotoxicity (Fig. 1). Cell lines derived from individuals of African ¶To whom correspondence should be addressed. E-mail: [email protected]. and European descent allowed us to define a set of genetic variants uchicago.edu. that contribute to chemotherapeutic-induced cytotoxicity through This article contains supporting information online at www.pnas.org/cgi/content/full/ their effects on gene expression in two different populations. The 0703736104/DC1. long-term goal is to identify gene polymorphisms that influence © 2007 by The National Academy of Sciences of the USA 9758–9763 ͉ PNAS ͉ June 5, 2007 ͉ vol. 104 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703736104 Downloaded by guest on October 2, 2021 Table 2. Biological process of GO for host and target genes CEU 35 YRI obtained from association studies of genotype and etoposide IC50 or of genotype and gene expression for combined, CEU, and YRI 25 Corrected Gene symbol* GO biological function P value 15 Determined from association analysis between genotype and IC50 FHOD3, FMN2 Cell organization and biogenesis 2 ϫ 10Ϫ4 Number of cell lines 5 GATA3, PCDH15 Sensory perception of sound 5.5 ϫ 10Ϫ3 DNM3, AP4S1 Endocytosis 5.9 ϫ 10Ϫ3 ϫ Ϫ3 0.6 0.8 1.0 1.4 1.6 1.8 FBN1, PCDH15, GRM8 Visual perception 6.6 10 0.4 1.2 2.0 ϫ Ϫ3 Box- Cox transformed etoposide IC50 FHOD3, FMN2 Actin cytoskeleton organization 6.6 10 and biogenesis Ϫ Fig. 2. Frequency distribution of Box–Cox transformed etoposide IC50 in the PCDH15, CDH2 Homophilic cell adhesion 7.7 ϫ 10 3 CEU (n ϭ 87) and YRI (n ϭ 89). DANJC6, PTPRD Protein amino acid 7.8 ϫ 10Ϫ3 dephosphorylation SPON1, PCDH15, CNTN5, HNT, Cell adhesion 8.3 ϫ 10Ϫ3 focus of this paper is to demonstrate our global genome approach CDH2 STX18, AP4S1 Intracellular protein transport 9.3 ϫ 10Ϫ3 in the context of identifying genetic variants important in response AP4S1, SORBS2, NUP205, Transport 9.7 ϫ 10Ϫ3 or toxicities associated with etoposide through their effect on gene SLC35C2 expression. KCNN3, SLC24A4 Potassium ion transport 9.9 ϫ 10Ϫ3 GATA3, KLF13 Transcription from RNA 0.01 Results polymerase II promoter Cell Cytotoxicity. Using the alamarBlue cytotoxicity assay, 87 and 89 KCNN3, GRM8 Synaptic transmission 0.01 ADCY2, FMN2, KSR2 Intracellular signaling cascade 0.01 cell lines derived from Center d’Etude du Polymorphisme Humain SPON1, FBN1, FMN2 Development 0.02 population (CEU) and Yoruban population (YRI) trios were KCNN3, SLIT1 Nervous system development 0.03 exposed to increasing concentrations of etoposide (0.02–2.5 ␮M) SLIT1, BMP7 Cell differentiation 0.03 for 72 h. Although our intention was to evaluate 90 CEU and 90 SLC24A4, GRID1 Ion transport 0.03 YRI lines, two CEU and one YRI cell lines failed to reach 85% Determined from association analysis between genotype and expression ϫ Ϫ4 viability on the experiment day on more than three attempts and CAPNS1, CAPN1, TCIRG1, Positive regulation of cell 2.3 10 POU3F2 proliferation therefore were not further evaluated. Additionally, one CEU cell TCIRG1, ATP13A1, ATP2A3 Proton transport 2.4 ϫ 10Ϫ4 line (GM12236) was not available from Coriell at the time of TUBA1, C9orf48 Microtubule-based movement 6.3 ϫ 10Ϫ3 phenotyping. Similar dose-dependent etoposide cell growth inhi- SLC27A4, ATP13A1, ATP2A3, Metabolism 7.6 ϫ 10Ϫ3 bition was observed in cell lines from both populations. Interindi- AGPAT2 ATP13A1, ATP2A3 Cation transport 0.01 vidual variation in the IC50 was 433- and 222-fold in CEU and YRI GBP2, GBP4, GBP7, NOTCH1 Immune response 0.01 cell lines, respectively (Fig. 2). The median IC50 in CEU and YRI ␮ IGSF8, ACTN4 Cell motility 0.01 cell lines was 0.43 and 0.40 M, respectively (5).
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