Computational protocol: Association of Topoisomerase II (TOP2A) and Dual-Specificity Phosphatase 6 (DUSP6) Single Nucleotide Polymorphisms with Radiation Treatment Response and Prognosis of Lung Cancer in Han Chinese

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Protocol publication

[…] To select candidate gene SNPs for genotyping, we searched the international HapMap database (http://hapmap.ncbi.nlm.nih.gov/) and identified sequences that included 1 kb upstream and downstream of each gene in SNP selection. We then downloaded genotype data restricted to those of the Han population from Beijing, China (CHB), and identified two SNPs (rs471692 in TOP2A and rs2279574 in DUSP6) with minor allele frequencies (MAF) above 1% and with a minimum value of 0.8 for the r2 parameter using the tagger algorithm implemented in Haploview Ver. 4.2 [,]. In particular, DUSP6 RS2279574 is localized in exon 1, and causes an amino acid change from Val to Leu, while TOP2A RS471692 is localized in an intron, which does not alter any amino acids of the TOP2A protein.To genotype these two SNPs, we collected 5 mL of peripheral blood from each participant into a sodium citrate tube, then extracted patient genomic DNA using a standard proteinase K digestion, followed by phenol-chloroform extraction and ethanol precipitation. These DNA samples were then genotyped using a TaqMan SNP genotyping assay kit (Affymatrix Inc., Cleveland, OH, USA) in an Applied Biosystems 7500 FAST Real-Time PCR System (Foster City, CA, USA). The primers and probes used for TOP2A rs471692 were 5′-ATC ACA AAC CTA AAA AAG AAA TCC A-3′ and 5′-AAT TAT TTA CCT GGG TCC ATG TTC T-3′; for DUSP6 RS2279574, 5′-AGC TTC TTG AGC AGCAGC CCG AGC A-3′ and 5′-CGA CTC GCC GCC CGT ATT CTC GTT C-3′. The TaqMan universal PCR master mix and predesigned SNP-genotyping assay mixture containing PCR primers and probes were purchased from ABI. The PCR amplification contained 25 μL master mix (Applied Biosystems), 10 μL of DNA, 2.5 μL of probe, and 12.5 μL of ddH2Or which was subjected to an initial denaturing step at 95°C for 10 minutes, followed by 47 cycles of 92°C for 30 seconds, 60°C for one minute, and a final extension at 60°C for one minute. To ensure accuracy of PCR amplification, we included three positive controls and two negative controls in each 96-well plate and randomly repeated 10% of the samples for quality control purposes. The concordance rate was 100% of the repeated analyses. A further 60 samples were randomly selected for direct DNA sequencing to confirm the TaqMan results, and DNA sequence data indicated 100% concordance. […]

Pipeline specifications

Software tools Tagger, Haploview
Application GWAS
Organisms Homo sapiens
Diseases Carcinoma, Non-Small-Cell Lung, Lung Neoplasms, Neoplasms