Copy number variation and cytidine analogue cytotoxicity: a genome-wide association approach

Abstract

Background: The human genome displays extensive copy-number variation (CNV). Recent discoveries have shown that large segments of DNA, ranging in size from hundreds to thousands of nucleotides, are either deleted or duplicated. This CNV may encompass genes, leading to a change in phenotype, including drug response phenotypes. Gemcitabine and 1-beta-D-arabinofuranosylcytosine (AraC) are cytidine analogues used to treat a variety of cancers. Previous studies have shown that genetic variation may influence response to these drugs. In the present study, we set out to test the hypothesis that variation in copy number might contribute to variation in cytidine analogue response phenotypes.

Results: We used a cell-based model system consisting of 197 ethnically-defined lymphoblastoid cell lines for which genome-wide SNP data were obtained using Illumina 550 and 650 K SNP arrays to study cytidine analogue cytotoxicity. 775 CNVs with allele frequencies > 1% were identified in 102 regions across the genome. 87/102 of these loci overlapped with previously identified regions of CNV. Association of CNVs with gemcitabine and AraC IC50 values identified 11 regions with permutation p-values < 0.05. Multiplex ligation-dependent probe amplification assays were performed to verify the 11 CNV regions that were associated with this phenotype; with false positive and false negative rates for the in-silico findings of 1.3% and 0.04%, respectively. We also had basal mRNA expression array data for these same 197 cell lines, which allowed us to quantify mRNA expression for 41 probesets in or near the CNV regions identified. We found that 7 of those 41 genes were highly expressed in our lymphoblastoid cell lines, and one of the seven genes (SMYD3) that was significant in the CNV association study was selected for further functional experiments. Those studies showed that knockdown of SMYD3, in pancreatic cancer cell lines increased gemcitabine and AraC resistance during cytotoxicity assay, consistent with the results of the association analysis.

Conclusions: These results suggest that CNVs may play a role in variation in cytidine analogue effect. Therefore, association studies of CNVs with drug response phenotypes in cell-based model systems, when paired with functional characterization, might help to identify CNV that contributes to variation in drug response.

Figure 1

Visualization of copy number regions identified in 15 randomly selected samples using Bead studio software. Randomly selected individual samples are listed on the X-axis and chromosomes on the Y-axis. Each colored bar represents one CNV call. Colors indicate copy number; where dark red indicates copy # 0, dark orange indicates copy # 1, dark blue indicates copy # 3, blue violet indicates copy # 4+. The thickness of the band indicates the length of the CNV region.

Figure 2

Comparison of chromosome 22 CNV regions identified using our 197 cell line samples compared with the results of previous studies in the UCSC genome browser. The Illumina 550 + 650 K (all samples combined), Illumina 550 K (CA, CEPH, HCA populations) and Illumina 650 K (AA samples) results in the diagram are from the present study, where spikes in the data indicate changes in CNV values. The "RefSeq Genes" row shows the locations of known genes in the human genome. In the "Structural Variation" tracks, green color indicates duplications, red indicates deletions, blue indicates both deletion and duplication, black represents an inversion and gray could be a gain or loss. "Conrad Dels" in the diagram are deletions detected by the analysis of SNP genotypes using the HapMap Phase I data, release 16c.1, CEU and YRI samples [11]. "Hinds Dels" are deletions observed during haploid hybridization analysis in 24 unrelated individuals from the Polymorphism Discovery Resource, selected for a SNP LD study [12]. "Iafrate CNVs" are from BAC microarray analysis of a population of 55 individuals [21]. "Locke CNVs" are CNV regions identified using array CGH in 269 HapMap individuals [22]. "McCarroll Dels" are deletions from genotype analysis, performed with HapMap Phase I data, release 16a [13]. "Redon CNVs" are from SNP and BAC microarray analysis of HapMap Phase II data [7]. "Sebat CNVs" represents oligonucleotide microarray analysis performed with a population of 20 normal individuals [7]. "Sharp CNVs" represents putative CNV regions detected by BAC microarray analysis in a population of 47 individuals [24]. The "Tuzun Fosmids" row consists of fosmid mapping sites detected by mapping paired-end sequences from a human fosmid DNA library [25].

Figure 3

Functional characterization for the SMYD3 candidate gene with specific siRNA knockdown. (A) Knockdown of the SMYD3 gene in human MIApaca-2 pancreatic cancer cells resulted in increased resistance to both AraC and gemcitabine as determined by MTS assay. SEM values for 3 independent experiments were so small that they are contained within the symbols. (B) Quantitative RT-PCR for SMYD3 in MIApaca-2 cells. Error bars represent SEM values for three independent experiments.