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Blood, Vol. 113, Issue 10, 2145-2153, March 5, 2009
Population-specific genetic variants important in susceptibility to cytarabine arabinoside cytotoxicity
Blood Hartford et al.
113: 2145
Supplemental materials for: Hartford et al
Association of levels of ara-CTP with DCK SNPs. The following 42 YRI cell lines were treated with 1mM ara-C for 6 hours: 12891, 18053, 18506, 18508, 18517, 18521, 18853, 18858, 18861, 18862, 18863, 18870, 18872, 19093, 19099, 19100, 19101, 19102, 19119, 19129, 19138, 19139, 19140, 19142, 19143, 19145, 19153, 19159, 19161, 19172, 19173, 19200, 19202, 19203, 19204, 19206, 19209, 19210, 19211, 19221, 19222, 19223. Cell pellets were spiked with 0.5mM guanosine diphosphate as an internal standard. Nucleotides were extracted using 125uL 0.25N PCA. After 30 minutes in an ice bath, the acid-soluble material was recovered by centrifugation (20,000 × g for 10 minutes at 4°C). The acid-soluble fraction was neutralized with 75uL 0.5N KOH. A Waters (Milford, MA) HPLC 2695 Alliance model equipped with a 996 diode array detector was used to quantitate the acid-soluble extracts. Samples of 200uL were injected onto a Partisil-10 SAX anion-exchange WCS analytical column (4.6mm × 250mm; Whatman, Inc., Clifton, N.J.) with a P-10 SAX solvecon precolumn (4.6mm × 250mm) and normal phase guard columns. The nucleotides were separated with a gradient of 50% Buffer A (0.005M (NH4)H2PO4 pH 2.8) and 50% Buffer B (0.5M (NH4)H2PO4 pH 2.8) to 100% Buffer B for 15 min followed by an isocratic elution of 100% Buffer B for 10 min at a flow rate of 1.5ml/min. The column was allowed to re-equilibrate to initial conditions for 5 min prior to the next injection. The absorbance of the nucleotides was determined at 280nm. A standard curve was calculated using a mixed population of lymphoblast cells spiked with araCTP concentrations ranging from 100 pmoles to 4 nmoles. The limit of detection was set at 100 pmole araCTP. The efficiency was 88%. Independent validation of phenotype, genotypes, and gene expression. The 49 CEU cell lines included in the validation experiments included: GM6986, GM6995, GM7002, GM7014, GM7016, GM7017, GM7049, GM7340, GM7341, GM7347, GM10837, GM10840, GM10841, GM10844, GM10845, GM10848, GM10858, GM11843, GM11891, GM11893, GM11894, GM11917, GM11918, GM11919, GM11920, GM11931, GM12045, GM12058, GM12096, GM12099, GM12116, GM12117, GM12718, GM12748, GM12749, GM12889, GM12890, GM13042, GM13044, GM13045, GM13046, GM13047, GM13048, GM13049, GM13050, GM13051, GM13052, GM13060, GM13133.
Files in this Data Supplement:
- Table S1. List of significant SNPs from the genotype-AUC QTDT association analysis in CEU population (PDF, 65.4 KB) -
P-values from genotype-expression and expression-AUC analyses are also listed.
- Table S2. List of significant SNPs from the genotype-AUC QTDT association analysis in YRI populations (PDF, 59.5 KB) -
P-values from genotype-expression and expression-AUC analyses are also listed.
- Figure S1. HPLC chromotogram identifiying ara-CTP in GM18858 6h after treatment with 1mM araC for 6h (dotted line) compared to no treatment (solid line) (JPG, 20.1 KB)
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Nucleotides were identified by their retention times and UV spectrums, in order of elution: GDP (RT = 7.4 min; λmax = 252.9); CTP (RT = 15.5 min; λmax = 277.8); araCTP (RT 17.1 min; λmax = 279.0); ATP (RT = 18.4 min; λmax = 255.2;); UTP (RT = 19.3 min; λmax = 260.0); TTP (RT = 20.7 min; λmax = 265.9); and GTP (RT = 23.67 min; λmax = 252.9.
- Figure S2. Association between cellular proliferation rate and AUC (JPG, 42.4 KB)
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(A) CEU cell lines (P<0.0001). (B) YRI cell lines (P<0.0001).
- Figure S3. Association between DCK mRNA expression and AUC (JPG, 34.5 KB)
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There was a significant relationship between DCK mRNA expression and AUC in both the CEU (r2 = 0.082, P=0.03) and YRI (r2 = 0.066, P=0.01) populations.
- Figure S4. Detection of DCK expression by immunoblot (JPG, 26 KB)
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Lysates from lymphoblastoid cells with different DCK mRNA level were resolved by 12% SDS-PAGE and transferred to PVDF membrane. The DCK expression was detected by probing with anti-DCK antibody and visualized by ECL reaction. Actin served as loading control.
- Figure S5. A three-step approach to identify genetic variants important in susceptibility to ara-C cytotoxicity (JPG, 35.1 KB)
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(A) In the first step a genotype-phenotype association was performed between HapMap SNP genotypes and ara-C cytotoxicity data from lymphoblastoid cell lines. (B) In the second step an association was performed between the significant SNPs identified in step 1 and gene expression data from the Affymetrix exon array. (C) In step three tests of association were performed between the significant genes identified in step 2 and ara-C cytotoxicity. The numbers in the triangle represent the number of SNPs significant for each step.
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