Large-scale evaluation of candidate genes identifies associations between VEGF polymorphisms and bladder cancer risk

Abstract

Common genetic variation could alter the risk for developing bladder cancer. We conducted a large-scale evaluation of single nucleotide polymorphisms (SNPs) in candidate genes for cancer to identify common variants that influence bladder cancer risk. An Illumina GoldenGate assay was used to genotype 1,433 SNPs within or near 386 genes in 1,086 cases and 1,033 controls in Spain. The most significant finding was in the 5' UTR of VEGF (rs25648, p for likelihood ratio test, 2 degrees of freedom = 1 x 10(-5)). To further investigate the region, we analyzed 29 additional SNPs in VEGF, selected to saturate the promoter and 5' UTR and to tag common genetic variation in this gene. Three additional SNPs in the promoter region (rs833052, rs1109324, and rs1547651) were associated with increased risk for bladder cancer: odds ratio (95% confidence interval): 2.52 (1.06-5.97), 2.74 (1.26-5.98), and 3.02 (1.36-6.63), respectively; and a polymorphism in intron 2 (rs3024994) was associated with reduced risk: 0.65 (0.46-0.91). Two of the promoter SNPs and the intron 2 SNP showed linkage disequilibrium with rs25648. Haplotype analyses revealed three blocks of linkage disequilibrium with significant associations for two blocks including the promoter and 5' UTR (global p = 0.02 and 0.009, respectively). These findings are biologically plausible since VEGF is critical in angiogenesis, which is important for tumor growth, its elevated expression in bladder tumors correlates with tumor progression, and specific 5' UTR haplotypes have been shown to influence promoter activity. Associations between bladder cancer risk and other genes in this report were not robust based on false discovery rate calculations. In conclusion, this large-scale evaluation of candidate cancer genes has identified common genetic variants in the regulatory regions of VEGF that could be associated with bladder cancer risk.

Figure 1. Gene Map and LD Plot of VEGF Gene

Color scheme is based on D′ and logarithm of the odds of linkage (LOD) score values: white D′ < 1 and LOD < 2, blue D′ = 1 and LOD < 2, shades of pink/red: D′ < 1 and LOD ≥ 2, and bright red D′ = 1 and LOD ≥ 2. Numbers in squares are D′ values (values of 1.0 are not shown). Block definition is based on the Gabriel et al. method [34]. Two (rs3024989 and rs367173) of the 29 SNPs determined are not shown because of low variation in this population. Red rectangles in the gene map represent exons.

Figure 2. Phylogenetic Trees for VEGF Haplotypes and Association with Bladder Cancer Risk among 926 Cases and 900 Controls with DNA in the iPLEX Assay, Spanish Bladder Cancer Study

See Figure 1 for block definitions. Of the 29 VEGF SNPs determined, two had low genotypic variation in our population; therefore, haplotype analyses were based on the remaining 27 SNPs. Polymorphic bases are in 5′ to 3′ order: Block 1(rs833052 and rs866236); Block 2 (rs1109324, rs1547651, rs833060, rs699947, rs1005230, rs833061, rs1570360, rs2010963, rs25648, rs833067, rs3025042, rs833068, rs3024994, rs735286, rs3024998, rs3025000, and rs3025006); and Block 3 (rs3025030, rs3025033, rs3025035, and rs3025036). Bolded rs numbers are for individual SNPs significantly associated with bladder cancer risk. Eleven cases and 13 controls with missing data on more than 15 of the 17 SNPs in Block 2 were excluded from haplotype analyses because their inclusion resulted in lack of convergence. Nucleotide changes significantly associated with risk in the individual genotype analyses are shown in boxes. The most common haplotye is the reference category. Haplotypes with the common variant for each individual SNP are CC for Block 1, GAGCCGTGCTGGCCCCC for Block 2, and GACC for Block 3.