The largest proportion of RCC cases were male (60.7%), from the Czech Republic (49.8%), and clear cell in histology (90%). Compared with controls, RCC cases had a higher BMI, were more probably to be exposed to lead, have self-reported hypertension and have a first-degree family history of cancer (). The difference in smoking status was no longer significant after adjusting for the other lifestyle and medical factors. Of the eight genes examined, only AGT had a statistically significant association with RCC risk (minimum P-value permutation test = 0.02) (). Similar statistical significance was achieved in association with three consecutive SNPs in the promoter region of the AGT gene using a 3-SNP sliding Haplowalk analysis (Haplowalk P = 0.02) (). Individual SNP analysis showed that at least one marker in the AGT (rs2493137: P = 0.001), AGTR1 (rs385338: P = 0.04) and SCNN1B (rs2303153: P = 0.04) genes was associated with RCC based on the test of trend adjusting for gender, age and country (). However, only the SNP in AGT remained statistically significant after taking multiple testing into account using the FDR (rs2493137: FDR-adjusted P = 0.02) (). Based on these gene-based findings, we focused our additional analyses and presented results for AGT.
Selected characteristics of participants
Hypertension-related genes and their association with RCC
HaploWalk and Haploview of the 17 SNPs examined in the AGT gene.
Of the 17 AGT
SNPs examined, five (29%) were significantly associated with RCC under an additive model adjusting for age, gender, country, smoking status, BMI, hypertension and lead exposure: rs1326889 (OR = 1.35, 95% CI = 1.15–1.58, P
-trend = 0.0002), rs2493137 (OR = 1.31, 95% CI = 1.12–2.54, P
-trend = 0.001), rs7539020 (OR = 1.22, 95% CI = 1.04–1.43, P
-trend = 0.02), rs3889728 (OR = 1.27, 95% CI = 1.07–1.51, P
-trend = 0.01) and rs3789662 (OR = 1.24, 95% CI = 1.03–1.49, P
-trend = 0.02) (). To determine which of these five AGT
SNPs was most strongly associated with cancer risk, we performed a backward elimination procedure including the five markers and the covariates listed above. Iteratively, the SNP with the highest P
-value was removed from the model. Using this approach, we determined that the kidney cancer association was largely driven by rs1326889 and secondly by rs2493137. The risk estimates in relation to each of these SNPs while adjusting for the effect of the other SNP were as follows: rs1326889 (OR = 1.26, 95% CI = 1.03–1.54, P
-trend = 0.02) and rs2493137 (OR = 1.12, 95% CI = 0.99–1.38, P
-trend = 0.05). Both of these SNPs are located in the promoter region of AGT
and are in high LD but only moderately correlated (D′ = 0.94, r2
= 0.40) (20
ORs and 95% CIs for RCC in relation to AGT SNPs
Stratified analysis suggested a modifying effect of hypertension and overweight/obesity on the genetic susceptibility of RCC risk in relation to AGT, although tests for interaction were not statistically significant (). Significant increased risks of RCC were seen in relation to the five AGT SNPs mentioned above (rs1326889, rs2493137, rs7539020, rs3889728 and rs3789662) in participants with either hypertension or a high BMI (≥25 kg/m2) or both of these factors, but there was no significant increased risk in participants without hypertension or with a normal BMI (<25 kg/m2). Among subjects with hypertension or a high BMI, the excess risks of RCC were again largely driven by rs1326889 (OR = 1.42, 95% CI = 1.20–1.70, P-trend = 0.001, P-interaction = 0.08) and rs2493137 (OR = 1.36, 95% CI = 1.14–1.63, P-trend = 0.001, P-interaction = 0.24). We also examined the associations stratified by hypertension status and BMI (<25 and ≥25 kg/m2) separately, but excess risk were not higher in just hypertensive or just overweight/obese subjects than what was observed in the main effect analysis. None of the five AGT SNPs associated with RCC were associated with either hypertension or high BMI among the controls. Approximately 93% of self-reported hypertensive subjects also reported using hypertension medication. Therefore, we did not have enough cancer cases to evaluate the heterogeneity of effect between subjects who took and did not take hypertension medication, but none of our findings changed measurably when subjects were limited to those who used medication. Associations between the AGT SNPs and RCC were not significantly modified by country, smoking status, lead exposure or histologic cell type (clear cell versus other subtypes).
ORs and 95% CIs for RCC in relation to AGT SNPs by hypertension and BMI
Strong LD was present among most of the 17 AGT
SNPs examined (). Among the five AGT
SNPs significantly associated with RCC, D′ ranged between 0.61 and 0.99 and r2
between 0.23 and 0.67 (20
). Based on the results of the Haplowalk analysis for AGT
, a region including five SNPs (rs1326889, rs7515609, rs2493137, rs7539020 and rs3889728) upstream of AGT
spanning intron 1 showed a strong association with RCC at an FDR of 0.05 (). We inferred five major haplotypes from this block with frequencies ranging between 7.3 and 45.7% among controls (). The global Score test for this block of haplotypes showed a statistically significant difference in the haplotype frequencies between cases and controls (P
= 0.02). Consistent with our single marker results, the haplotypes, T-T-T-T-G and T-C-T-T-G, that contain the risk-conferring alleles of rs1326889, rs2493137, rs7539020 and rs3889728, were significantly associated with 1.37- and 1.51-fold risks of RCC compared with the common haplotype (C-T-C-C-A) (). These two haplotypes were significantly associated with RCC among participants with either hypertension or a high BMI (T-T-T-T-G: OR = 1.48, 95% CI = 1.17–1.88; T-C-T-T-G: OR = 1.46, 95% CI = 1.01–2.09) (global P
= 0.003), but not among those without both of these factors compared with the common haplotype (global P
= 0.49) (). As with the individual SNPs, these haplotypes were not associated with hypertension or BMI in the controls.
ORs and 95% CIs for RCC in Relation to AGT haplotypes