Mapping genes that contribute to daunorubicin-induced cytotoxicity

Abstract

Daunorubicin is an anthracycline antibiotic agent used in the treatment of hematopoietic malignancies. Toxicities associated with this agent include myelosuppression and cardiotoxicity; however, the genes or genetic determinants that contribute to these toxicities are unknown. We present an unbiased genome-wide approach that incorporates heritability, whole-genome linkage analysis, and linkage-directed association to uncover genetic variants contributing to the sensitivity to daunorubicin-induced cytotoxicity. Cell growth inhibition in 324 Centre d' Etude du Polymorphisme Humain lymphoblastoid cell lines (24 pedigrees) was evaluated following treatment with daunorubicin for 72 h. Heritability analysis showed a significant genetic component contributing to the cytotoxic phenotypes (h2 = 0.18-0.63 at 0.0125, 0.025, 0.05, 0.1, 0.2, and 1.0 mumol/L daunorubicin and at the IC50, the dose required to inhibit 50% cell growth). Whole-genome linkage scans at all drug concentrations and IC50 uncovered 11 regions with moderate peak LOD scores (> 1.5), including 4q28.2 to 4q32.3 with a maximum LOD score of 3.18. The quantitative transmission disequilibrium tests were done using 31,312 high-frequency single-nucleotide polymorphisms (SNP) located in the 1 LOD confidence interval of these 11 regions. Thirty genes were identified as significantly associated with daunorubicin-induced cytotoxicity (P < or = 2.0 x 10(-4), false discovery rate < or = 0.1). Pathway and functional gene ontology analysis showed that these genes were overrepresented in the phosphatidylinositol signaling system, axon guidance pathway, and GPI-anchored proteins family. Our findings suggest that a proportion of susceptibility to daunorubicin-induced cytotoxicity may be controlled by genetic determinants and that analysis using linkage-directed association studies with dense SNP markers can be used to identify the genetic variants contributing to cytotoxicity.

Figure 1

Boxplots for 324 cell lines within 24 pedigrees are shown for various concentrations of daunorubicin, illustrating interfamily and intrafamily variance. The mean for each family’s percentage survival after daunorubicin treatment for 72 h with (A) 0.0125 µmol/L; (B) 0.025 µmol/L; (C) 0.2 µmol/L, and (D) 1.0 µmol/L. Line, mean phenotypic response within each family; box, mean ± SE; whiskers, mean ± 1.97 × SE.

Figure 2

Linkage-directed association studies on chromosome 4 to identify SNPs conferring sensitivity to daunorubicin-induced cytotoxicity. Top, results from linkage analysis based on 24 CEPH families. The 1 LOD confidence interval of the peak on chromosome 4 from multipoint linkage of daunorubicin-induced cytotoxicity (0.05 µmol/L: black solid curve) and from the linkage analysis of 0.1 µmol/L, IC₅₀, 0.2 µmol/L, 0.025 µmol/L, and 0.0125 µmol/L(dotted curves with peaks in descending order). Vertical dashed lines, 1 LOD confidence interval of the linkage peaks for the follow-up association studies; horizontal dashed lines, P value of 2 × 10⁻⁴. Results of QTDT analysis using genotypes for 30 CEU trios from the HapMap Project (gray bars) and associated genotypes within INPP4B (black bars). Middle, results of QTDT analysis illustrating associated genotypes within INPP4B. Bottom, SNP (rs978752) located in the 16th intron of INPP4B gene shows suggestive association evidence with daunorubicin (1 µmol/L)–induced cytotoxicity (FDR = 0.66, P = 3 × 10⁻⁵).This SNP is also modestly associated with other daunorubicin phenotypes (0.1 µmol/L: FDR = 0.1, P = 2 × 10⁻⁴;0.2 µmol/L: FDR = 0.22, P = 4 × 10⁻⁵).

Figure 3

Linkage-directed association studies on chromosome 16 to identify SNPs conferring sensitivity to daunorubicin-induced cytotoxicity. Top, results from linkage analysis based on 24 CEPH families. The 1 LOD confidence interval of the peak on chromosome 16 from multipoint (solid curve) linkage of daunorubicin (0.0125 µmol/L)–induced cytotoxicity. Vertical dashed lines, 1 L OD confidence interval of the linkage peaks for the follow-up association studies; horizontal dashed lines, P value of 2 × 10⁻⁴. Results of QTDT analysis using genotypes for 30 CEU trios from the HapMap Project (gray bars) and associated genotypes within CDH13 (black bars). Middle, results of QTDT analysis illustrating associated genotypes within CDH13. Bottom, SNP rs1862831 in the fifth intron of CDH13 gene are associated with daunorubicin (0.0125 µmol/L)–induced cytotoxicity (FDR = 0.03, P = 1 × 10⁻⁶).