Genotype Panel

Background

Variants in numerous genes are thought to affect the success or failure of cancer chemotherapy. Interindividual variability may be due to genes involved in drug metabolism and transport, drug targets (receptors, enzymes, etc.), and proteins relevant to cell survival (eg, cell cycle, DNA repair, and apoptosis). The purpose of the current study is to establish a flexible, cost-effective, high-throughput genotyping platform for candidate genes involved in chemoresistance and sensitivity and treatment outcomes.

Methods

We have adopted SNPlex for genotyping of 432 single nucleotide polymorphisms (SNPs) in 160 candidate genes involved in response to cancer chemotherapy.

  • SNPlex Reagent and Probe Sets

For the selected SNPs, NCBI SNP reference group IDs (rs numbers) or SNP sequences were submitted to Applied Biosystems for the design of SNPlex panels following their proprietary selection algorithms. We separated genes into different groups: drug metabolism and transport, DNA repair/apoptosis, and cell cycle/cell growth/drug targets. The DNA sequence surrounding a specific polymorphism must meet specific requirements for probe design, including but not limited to: A. genomic screening; the DNA sequence flanking the target SNP must be unique and have no more than 1 genomic alignment match with 21 or more contiguous bases to ensure hybridization specificity, and there is no second nearby SNP. B. The target sequences must have adequate characteristics for annealing efficiency. C. Grouping Rules: Strict grouping rules are used to determine the optimal multiplex composition. SNPlex panels and reagents were synthesized by Applied Biosystems.

  • DNA samples

Thirty-nine blood DNA samples from chronic lymphocytic leukaemia patients were collected by Dr John Byrd following institutional review board (IRB) protocol at The Ohio State University for phase I clinical trials of flavopiridol at the Comprehensive Center of the Ohio State University Cancer. In addition, 90 colorectal cancer specimens were chosen from a series of 1262 consecutively pooled patients with colorectal carcinoma diagnosed at major hospitals in the Columbus metropolitan area, whose tumours did not show microsatellite instability, as previously described. Control groups were obtained from previously genotyped cohorts where the same SNPs can be accessed (HapMap and other data sets as indicated). The research protocol and consent form were approved by the institutional review board of each participating hospital, and all patients provided written informed consent.

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  • Genotyping of the UGT1A1 promoter dinucleotide repeat polymorphism (TA) nTAA

The UGT1A1 dinucleotide repeat was genotyped according to previously designed PCR sequences and PCR conditions [32]. The forward primer sequence was 5′-GTCACGTGACACAGTCAAAC-3′. The reverse primer sequence was 5′-TTTGCTCCTGCCAGAGGTT-3′ and was labelled with FAM. The PCR products were analyzed using an Applied Biosystems 3730 DNA Analyzer.

  • Data analysis

Hardy-Weinberg equilibrium for each SNP was analyzed using HelixTree according to the manufacturer’s manual (Golden Helix, Inc. Bozeman, MT, USA).

Results

Genotype panels were applied to 39 patients with chronic lymphocytic leukaemia undergoing chemotherapy with flavopiridol and 90 patients with colorectal cancer. 408 SNPs (94%) produced successful genotyping results. Additional genotyping methods for polymorphisms undetectable by SNPlex were established, including multiplexed SNaPshot for CYP2D6 SNPs and PCR amplification with fluorescently labelled primers for the UGT1A1 (TA) nTAA promoter repeat polymorphism.

Conclusion

This genotyping panel is useful in supporting clinical trials of anticancer drugs to identify polymorphisms that contribute to interindividual variability in drug response. The availability of population genetic data across multiple studies has the potential to generate genetic biomarkers to optimize cancer therapy.