Genes and polymorphisms selected for genotyping by SNPlex
We have designed cancer genotyping SNPlex panels, selecting genes involved in drug metabolism and transport, DNA repair and apoptosis, cell cycle/cell growth/drug targets. We have selected polymorphisms for genotyping along the following criteria: polymorphisms known to affect enzyme/transporter functions, and SNPs in transcribed genic regions and htSNPs with high abundance obtained from HapMap and other databases. We have selected 560 SNPs for 160 genes, ordered into different categories:
Transporters: ABCA1, ABCA2, ABCA3, ABCA9, ABCA10, MDR1/ABCB1, ABCB4, ABCB11, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC6, ABCG2/BCRP, ABCG5, ABCG8, SLC19A1 (RFC) and SLC21A6.
Phase I metabolism enzymes: CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, CYP2E1, 3A4, 3A5, 17A1, DIA4/NQO1, EPHX1/EH, MPO and SOD2.
Phase II metabolism enzymes: GSTA1 GSTA2, GSTA4, GSTM1, GSTM3, GSTP1, GSTT1, GSTT2, NAT1, NAT2, SULT1A1, SULT1A2, TPMT, COMT, UGT1A1, UGT1A6, UGT1A7, UGT1A9 and UGT2B7.
DNA repair genes: ADPRT/PARP, ADPRTL1, APEX1/APE1, ATM, ATR, BARD1, BLM, BRCA1, BRCA2, CHEK2, ERCC2/XPD, ERCC4/XPF, ERCC5/XPG, FANCD2, LIG1, LIG3, LIG4, MGMT/AGT, MLH1, MPG, MSH2, MSH3, MSH6, MYH/MUTYH, NBS1, NT5E, OGG1, PCNA, PMS2, POLB, RAD23A, RAD51, RAD52, RAD54B, RAD9A, RECQL, WRN, XPA, XPC, XRCC1, XRCC2, XRCC3, XRCC4, XRCC5 and XRCC9/FANCG.
Drug targets, cell signaling, cell cycle and apoptosis related genes: DHFR, DPYD, TYMS, VKORC1, EGFR, ERBB2, FLT1 (VEGFR1), KDR (VEGFR2), FLT4 (VEGFR3), PDGFRA, PDGFRB, KIT, RET, CDA, BAX, CASP3, CASP8, CASP9, CASP10, CCND1, CCNH, CDK7, CDKN1A/p21, CDKN1B/p27, CDKN2A/p16, CDKN2B/p15, GADD45A, IRS2, MDM2, RB1, TERC/hTR, TERT, TP53, TP53BP1, TP53BP2, TP73, APC, NF1, NF2, HPC1, VHL, ECRG1, WT1, MEN1, SMAD2, SMAD4, TNFRSF10A, PTCH and CDH1.
Among the 560 SNPs, 432 SNPs (77%) were successfully designed to be included in the SNPlex panels [see Additional file 1
]. The SNPs were divided into several groups so that a subset of the SNPlex panels might be sufficient for a specific research project.
• Drug metabolism and transports: 4 panels, 189 SNPs.
• DNA repair: 3 panels, 148 SNPs.
• Cell cycle/growth/apoptosis: 2 panels, 95 SNPs.
The selection of polymorphisms for this study included some redundancy to account for limitation of the SNPlex approach. Any polymorphisms that could not be included with the SNPlex panels were omitted, or if thought to be critical, targeted by alternative methods. For example, a majority of the SNPs that are not suitable for SNPlex genotyping can be genotyped by multiplexed SNaPshot assay (see multiplexed SNaPshot for CYP2D6
in this manuscript as an example). Similarly, small insertions/deletions and repeats can be amplified by PCR and the variants determined by PCR product size difference based on gel electrophoresis or capillary electrophoresis using fluorescent-labeled primers (see UGT1A1
promoter dinucleotide repeat polymorphism in this manuscript). Based on the sequence information and literature search, possible alternative methods for detection of these genetic variants are listed in Additional file 1