Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.
BMC Genomics. 2010 Jun 4;11:357. doi: 10.1186/1471-2164-11-357.
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.
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.
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.
人类基因组显示出广泛的拷贝数变异（CNV）。最近的发现表明，大小从数百到数千个核苷酸的大片段 DNA 被删除或复制。这种 CNV 可能包含基因，导致表型变化，包括药物反应表型。吉西他滨和 1-β-D-阿拉伯呋喃糖基胞嘧啶（AraC）是用于治疗多种癌症的胞嘧啶类似物。先前的研究表明，遗传变异可能影响对这些药物的反应。在本研究中，我们着手测试假设，即拷贝数的变化可能导致胞嘧啶类似物反应表型的变化。
我们使用了一个基于细胞的模型系统，该系统由 197 个具有种族定义的淋巴母细胞系组成，使用 Illumina 550 和 650 K SNP 阵列获得了全基因组 SNP 数据，用于研究胞嘧啶类似物的细胞毒性。在基因组的 102 个区域中发现了 775 个等位基因频率> 1%的 CNV。102 个这些基因座与先前鉴定的 CNV 区域重叠。与吉西他滨和 AraC IC50 值相关的 CNV 鉴定出 11 个具有置换 p 值< 0.05 的区域。进行多重连接依赖性探针扩增测定以验证与该表型相关的 11 个 CNV 区域；对于计算机发现的假阳性和假阴性率分别为 1.3%和 0.04%。我们还对这些相同的 197 个细胞系进行了基础 mRNA 表达数组数据，这使我们能够量化鉴定出的 CNV 区域内或附近的 41 个探针的 mRNA 表达。我们发现，在我们的淋巴母细胞系中，这 41 个基因中有 7 个基因高度表达，并且在 CNV 关联研究中具有显著性的 7 个基因之一（SMYD3）被选择用于进一步的功能实验。这些研究表明，在细胞毒性测定中，SMYD3 的敲低增加了胰腺癌细胞系对吉西他滨和 AraC 的耐药性，这与关联分析的结果一致。
这些结果表明，CNV 可能在胞嘧啶类似物作用的变化中起作用。因此，在基于细胞的模型系统中，CNV 与药物反应表型的关联研究与功能表征相结合，可能有助于鉴定导致药物反应变化的 CNV。
