Copy number variation and cytidine analogue cytotoxicity: a genome-wide association approach.
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.
Kalari
Krishna R
KR
Division of Clinical Pharmacology, Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA. weinshilboum.richard@mayo.edu
Hebbring
Scott J
SJ
Chai
High Seng
HS
Li
Liang
L
Kocher
Jean-Pierre A
JP
Wang
Liewei
L
Weinshilboum
Richard M
RM
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence; D002460:Cell Line; D002472:Cell Transformation, Viral; D003561:Cytarabine / Q000494:pharmacology*; D056915:DNA Copy Number Variations / Q000235:genetics*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D055106:Genome-Wide Association Study; D004854:Herpesvirus 4, Human / Q000235:genetics; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D008297:Male; D021141:Nucleic Acid Amplification Techniques; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D000093542:Gemcitabine
D000970:Antineoplastic Agents; D003561:Cytarabine; D003841:Deoxycytidine; D011495:Histone-Lysine N-Methyltransferase; C506278:SMYD3 protein, human; D000093542:Gemcitabine